INDIAN ASSOCIATION OF MEDICAL MICROBIOLOGISTS

A.P. CHAPTER

BULLETIN OF IAMM AP CHAPTER

Website : http://sites.google.com/site/iammapchapter/

Blog : http://iammapchapter.blogspot.com/ Email : iammapchapter@yahoo.co.in

January 2014 Vol. 17(1) Editor : Dr. D.S.Murty

President’s Message From the Secretary’s Desk

A happy new year and Pongal to all. The past year

has been a memorable one with the national

conference hosted by NIMS HYDERABAD.2014 is also

going to be special with the state conference being

held in the first week of the first month at Kamineni

Institute of medical sciences.

The challenge is always there between the seen and

the unseen, and we are trying our best to strike a

positive balance. Let us update our knowledge and

strive to train our enthusiastic postgraduates as

clinical microbiologists.

Thanking you all,

With regards and best wishes,

Dr. I. Jyothi PadmajaPresident, IAMM AP Chapter,

Professor and Head of Microbiology, Andhra Medical College,

Visakhapatnam

INSIDE

::: SAVE PAPER – SAVE NATURE – VISIT WEBSITE FOR UPDATES :::

Published by : Dr. K.Prasanthi, Secretary on behalf of IAMMAP Chapter

Dear Members, Warm welcome to the 2014 annual conference of IAMM AP chapter . The journey to Kamineni medical college, in a pleasant chilling weather of January , leaving behind the pollution , dreaming the newer challenges in clinical Microbiology , definitely gives a better hope for future microbiologists. You all know that the trend has changed from Laboratory to Clinical Microbiology , which redefines our job descriptions and entailing several changes in education, training and practice . Among the new demands , rapidity is the need of the hour which insist clinical microbiology labs to choose advanced technologies and new methods .

This again impose labs to train and develop new skills to meet the requirements . Attending CME , workshops and annual conferences provide such platform to improve our knowledge.But remember , leaving the laboratory and going into the clinical areas does not mean the end of basic science, ratherthe basic microbiology results based on microscopy, culture or serology always play a value in patient care. With Best wishes...

Dr.K.PrasanthiSecretary – IAMM AP Chapter

Associate Professor of Microbiology,Gandhi Medical College, Secunderabad.

Email : beboldprasanthi90@gmail.com

MICROCON 2014 - page 2

Executive body - page 2

Microbiology news - page 3

Recent advances in the diagnosis of Mycobacterium

tuberculosis - page 4

Identifying clinically important anaerobes- a

demanding task - page 8

Fluorescence Microscopy - page 18

Preservation of fungi - page 23

MDRTB-A synopsis - page 26

HIV Testing strategies - page 29

MICROCON 2014

The 38th

National Conference of the Indian Association of Medical Microbiologists to be

hosted from 15th

- 19th

October 2014 at Birla Auditorium Jaipur, Rajasthan, India.

The conference theme is “Syndromic & Integrative Approach - The Way Ahead”.

Last Date For Early Bird Registration is 31st January 2014.

Abstract Submission deadline 15th July 2014.

WEBSITE for details : www.microcon2014.com

The registration form and first information brochure are also available on the website of

IAMMAPChapter.

THE EXECUTIVE COUNCIL OF IAMM AP CHAPTER

President : Dr. I. Jyothi Padmaja,

Professor and Head of Microbiology, Andhra Medical College, Visakhapatnam

Vice President :

Dr. P.R.Anuradha, Professor and head of Microbiology,

Gandhi Medical College, Secunderabad

Secretary : Dr. K. Prasanthi,

Gandhi Medical College, Secunderabad

Ph : 9490119539

Treasurer : Dr. P. Venkataramana,

Asst. professor of Microbiology, Andhra Medical College, Visakhapatnam

Ph : 9885170050

Joint Secretary : Dr. D.S.Murty,

Asst. Professor of Microbiology, Osmania Medical College, Hyderabad

Ph : 9440341099

EXECUTIVE MEMBERS :

Dr. K.Saileela,

Professor and Head of Microbiology,

Kaimineni Institute of Medical Sciences, Narketpally

Dr. P.Sreenivasulu Reddy,

Professor of Microbiology,

Narayana Medical College,

Dr. R.Sankara Rao,

Asst Professor of Microbiology,

Andhra Medical College,

Dr. P. Kasturi,

Asst. Professor of Microbiology,

Rangaraya Medical College,

Associate Professor of Microbiology,

Email : beboldprasanthi90@gmail.com

Bulletin of IAMM AP Chapter 2 Vol.17(1)

Nellore

Visakhapatnam

Kakinada

MICROBIOLOGY NEWS :

From Friend to Foe: How Benign Bacteria

Evolve Into Virulent Pathogens :

Dec. 12, 2013 — Bacteria can evolve rapidly to

adapt to environmental change. When the

"environment" is the immune response of an

infected host, this evolution can turn harmless

bacteria into life-threatening pathogens. A study

published on December 12 in PLOS Pathogens

provides insight into how this happens.

Isabel Gordo and colleagues from the Instituto

Gulbenkian de Ciencia in Oeira, Portugal, have for

the first time devised an experimental system to

observe and study the evolution of bacteria in

response to encounters with cells of the

mammalian immune system. They found that in

less than 500 bacterial generations (or 30 days),

the bacteria became more resistant to being

killed by immune cells and acquired the ability to

cause disease in mice.

"Escherichia coli bacteria show an extraordinary

amount of diversity: Many are benign commensal

bacteria, but some are deadly pathogens," says

Isabel Gordo. "It is thought that many strains of E.

coli that cause disease in humans evolved from

commensal strains. We thought that

experimental evolution would be a powerful tool

to directly observe some of the steps E. coli may

take in the transition from commensalism to

pathogenesis."

For their study, the scientists studied initially

benign E. coli bacteria that were continuously

confronted with macrophages, which are part of

our immune system and can swallow and digest

bacteria. They grew a mix of bacteria and

macrophages in a liquid culture (a glass bottle

that contains a nutritious broth). Once a day, they

diluted the mix, and every other day they took a

sample of the bacteria for further analysis. As a

control, they grew, diluted, and analyzed bacteria

from the same ancestral strain but grown without

macrophages.

From day four on, bacteria that had been exposed

to macrophages started to show changes in their

phenotype (their appearance), whereas such

changes were never observed in the controls. The

selective pressure imposed by the presence of the

macrophages prompted changes in the bacteria

that were consistently observed in six

independent experimental series. The changes

affected the phenotype of the bacteria (with new

variants forming either "small colonies" or

"mucoid colonies"), their fitness, and their genetic

make-up.

When the scientists looked at the interaction

between new variant bacteria and macrophages

more closely, they found that the small colony

variants were more resistant to being digested by

macrophages than the ancestral strain, and the

mucoid variant was less likely to be gobbled up.

When they infected mice with mucoid variant

bacteria, they also found that the variants have

increased ability to cause disease in mice.

"We demonstrate," the scientists say, "that E. coli

can adapt to better resist macrophages within a

few hundred generations, and that clones with

morphologies and traits similar to those of

pathogenic bacteria rapidly emerge."

Source : Public Library of Science (2013, December 12).

From friend to foe: How benign bacteria evolve into

virulent pathogens. ScienceDaily. Retrieved December

16, 2013, from http://www.sciencedaily.com

/releases/2013/12/131212185831.htm

Bulletin of IAMM AP Chapter 3 Vol.17(1)

Recent Advances in Rapid Laboratory Diagnosis of MycobacteriumTuberculosis

Dr. K.R.L.Surya Kirani, Professor of Microbiology,

Rangaraya Medical College, Kakinada INTRODUCTION

The bedside decision on the initiation of anti-tuberculous drug therapy are based on epidemiologic, clinical, radiographic, and/or histological findings, supported by a rapid microbiologic test, a positive acid-fast bacilli (AFB) smear result.

Before the introduction of molecular biology into diagnostic mycobacteriology, direct microscopy using a Ziehl-Neelsen smear of early morning sputum was the only way of making a rapid diagnosis. This requires the presence of about 104 acid-fast bacilli (AFB) per ml of sputum. The sensitivity could be improved by concentrating sputum ,applying auramine O fluorescent stain. Direct microscopy cannot distinguish between M.tuberculosis and nontuberculous mycobacteria.

Molecular diagnostics in tuberculosis have enabled

(i) direct detection of M. tuberculosis complex in clinical specimens;

(ii) identification of mycobacteria, (iii) detection of drug resistance of M.

tuberculosis, (iv) DNA typing to address issues like

reactivation of disease versus exogenous reinfection, and to track transmission and internal laboratory contamination.

(v) typing for epidemiological investigation

These technological advancements are not intended to replace the conventional tests, but would rather serve as important complementary tools in the management of tuberculosis.

Comparison of turnaround time and sensitivity for laboratory diagnosis of Mycobacterium tuberculosis :

Method Time Sensitivity

AFB smear 2 hours < 50%

LJ culture 2-8 weeks

MGIT 960 culture 1-6 weeks

Mycobacterial culture identification&drug susceptibility testing 2-3 weeks

Molecular Diagnosis 4 days

BDProbeTecTM 4-5 hours 86-92%

GenProbe AMTDTTM 4-6 hours 85-95%

ROCHE COBASTM 5-6 hours 78-97%

In-house IS6110 PCR 18 hours 88-94%

rpoB PCR sequencing forRIF resistance 72 hours 92%

GENOMICS

Genome of M.tb is a Single large circular DNA

molecule ,G+C content 60-70mol%.

One copy of genes encoding ribosomal

RNA.Genome sequence of M.tb H37Rv has 4000

genes encoding proteins,50 genes for RNAs.One

single copy of RNA operon located 1500kb from

the origin of replication resulting in slow

growth.Rec A gene is unusual in that it encodes

an 85kDa protein though RecA protein is

only38kDa.Genes involving aerobic and anerobic

metabolism are present to enable survival in

granulomas/oxygen poor tissues.

In 9% genes ,sequences belong to—Pro-

Glu(PE),Pro-Pro-Glu(PPE)—Gene

Bulletin of IAMM AP Chapter 4 Vol.17(1)

families.(Glycene rich proteins families-)leading

to genetic variability. This is the source of

antigenic variation/interference with host

immune responses by inhibiting antigen

processing.

Rich in repeated DNA sequences—Insertion

Sequences(IS),tandem repeats inserted in

intercistronic regions of genes within an operon

(MIRU) Mycobacterial Intersperced Repetitive

Units

H37Rv—56IS from eight different families have

been identified.IS6110—wide range of copy

numbers and high polymorphism within

copy.H37Rv—MIRU in 41 loci display

polymorphism—used for strain typing.

The polymorphism within the kat G gene and

gyrA gene identifies three genetic groups.First

encompasses all tubercle bacilli including M.tb,

M.bovis,M.africanum,M.microti.Other two

include M.tb only.

Resistance is genetically controlled. Detection of

resistance genes is the advanced lab test using

Gene Probes ,Molecular methods .

The genetic basis of antituberculous drug

resistance through identification of the main

genes –

rpoB (rifampicin), katG(isoniazid), inhA (isoniazid and ethionamide), ahpC (isoniazid), pncA (pyrazinamide),embB (ethambutol), rrs (streptomycin), rpsL (streptomycin) and gyrA14(fluoroquinolone). No plasmids in M.tb.

MOLECULAR METHODS FOR DETECTION

The primary determinant of successful

NAA(Nucleic acid assay) testing of tuberculosis

depends on the shedding of mycobacterial DNA

in secretions from caseating granuloma and its

dissemination into sterile body fluids or tissue

biopsies. In multibacillary diseases with high

mycobacterial load, a positive Ziehl-Neelsen (ZN)

smear with positive NAA is diagnostic of active

tuberculosis.A positive ZN smear with a negative

NAA in the absence of inhibitors would indicate

non-tuberculous mycobacterial diseases.

Gene amplification methods for direct detection

of M.tuberculosis sequences from clinical

specimens classified as those based on

polymerase chain reaction (PCR) and isothermal

amplification reactions .

Gene amplification techniques are highly

sensitive and under optimum conditions may

detect 1-10 organisms. These assays can play a

very useful role in early confirmation of diagnosis

in paucibacillary extra-pulmonary forms of

tuberculosis.The role of NAA test is more

important in paucibacillary diseases with low

mycobacterial load. But the presence of PCR

inhibitors-to polymerase enzyme- especially in

extrapulmonary specimens may produce false

negative results. A brief culture augmentation

step before NAA to enhance the mycobacterial

load with concomitant dilution of inhibitors can

maintain the sensitivity.

Several systems exist for the rapid identification

of mycobacterial species from cultured isolates .

The MicroSeq 500 system (Applied Biosystems,

Foster City, California,US) is an assay based on

sequencing a portion of the 16S rDNA gene .

The ACCUPROBE M.tuberculosis

Principle of the Test

Nucleic acid hybridization tests are based on the

ability of complementary nucleic acid strands to

specifically align and associate to form stable

double-stranded complementary to the

ribosomal RNA of the target organism. After the

ribosomal RNA is released from the organism, the

labeled DNA probe combines with the target

organism’s ribosomal RNA to form a stable DNA:

RNA hybrid.It is a rapid DNA probe test which

utilizes the technique of nucleic acid

hybridization complexes.

The ACCUPROBE SYSTEM uses a single-stranded

DNA probe with a chemiluminescent label that is

for the identification of Mycobacterium

tuberculosis complex and MOTT from culture.

Bulletin of IAMM AP Chapter 5 Vol.17(1)

The Selection Reagent allows for the

differentiation of non-hybridized and hybridized

probe. The labeled DNA: RNA hybrids are

measured in a GEN-PROBE luminometer. A

positive result is a luminometer reading equal to

or greater than the cut-off. A value below the

cut-off is a negative result.

SPOLIGOTYPES-spacer oligotyping

Polymorphism of direct repeat(DR) locus specific

for M.tb complex.Contains multiple conserved

36bp repeats interspersed with non repetitive

short spacer sequences of 34-41 bp length.Tested

by hybridising their PCR amplified DR region to a

membrane that consists of an array of 43

covalently bound oligonucleotides representing

the polymorphic spaces identified with M.tb

H37Rv DR sequence.Absence of spaces 33-36—

M.tb spoligotypes

DNA probes: defined oligonucleotide probes for

identification of M. tuberculosis, M. avium and

several other mycobacteria. When used along

with newer methods of detection of the early

growth (such as BacT Alert 3D, Septi-check, MGIT

etc) help in rapidly confiriming the diagnosis than

with classical biochemical tests.

Species identification-- 16S &23S rDNAS ,gyrB

gene,dnaJgene,16S-23S rRNA spaces gene,hsp 65

gene.,mtp40 gene-phospholipaseC is in M.tb

only,pncA &oxy R genes in bovis and M.tb.

Ribosomal rRNA based probes: target rRNA,,

ribosomal DNA,spacer and flanking sequences for

quick identification. earlier radiolabeld,

developed into chemiluminescent techniques

10-100 fold more sensitive than DNA targeting to

confirm the diagnosis directly in the clinical

specimens in a good proportion of cases.The

lowest detection limit is around 100 organisms.

Gene amplification methods for identification:

Amplification of specific gene regions followed by

hybridization with species specific probes,

sequencing and RFLP analysis -- hsp 65 kDa gene,

katG and rRNA genes have been described. These

PCR-RFLP assays help in quick identification from

the culture isolates, directly from the clinical

specimens.

DNA strip-12 assays based on the reverse

hybridization of PCR products to oligonucleotide

probes bound on a membrane strip can be

applied to identify mycobacterial species.

Inno-LiPA Mycobacteria (Innogenetics NV, Ghent,

Belgium) by the 16 to 23S rDNA spacer region of

Mycobacterium species (Inno-LiPA Mycobacteria

v2)by increasing the number of identifiable

mycobacterial species to 16.

GenoType Mycobacteria (Hain Lifescience,

Nehren, Germany)by 23S rDNA of

Mycobacterium species . can identify 13 clinically

important mycobacterial species from clinical

isolates or broth culture systems , but does not

allow differentiation among members of the M.

tuberculosis complex

DRUG RESISTANCE

Orange G3TB tuberculosis –

Total assay time: 4 hours

Steps : Sample (Sputum,CSF) Biological

inactivation DNA Gene

amplification Fluorometric detection

The line probe assay (INNO –LiPA Rif TB,

Innogenetics Ghent, Belgium), is a commercially

available reverse hybridization – based probe

assay for rapid detection of rifampin mutations

leading to rifampin resistance in M.tuberculosis.

Consists of Internal transcribed Spacer(ITS) region

of 280 bp which separates the 16S &23S

Bulletin of IAMM AP Chapter 6 Vol.17(1)

rDNAS.Some kits contain specific alleles of the

gyrB gene probes.

INNO-LiPA Rif.TB(Innogenetics, Belgium) and

MisMatch Detect II (Ambion, U.S.A.) have

performed well for direct detection of rifampicin

and isoniazid resistance rapidly in clinical

specimens.

ADVANCES

The peptide nucleic acids probes as an, in situ 13

hybridization assay by a fluorescent stain

format.Peptide nucleic acids are DNA-like

structures in which the sugar-phosphate

backbones are replaced with peptide-like

structures. The binding of peptide nucleic acid to

DNA is sequence specific, and the interaction is

stronger than that of a DNA-DNA

interaction.Utility of the peptide nucleic acid

probes is in detecting mutated KatG and rpoB

genes.

Molecular beacons are molecules that emit light

following upon a chemical reaction involving a

colored fluorophore.Molecular beacon assays

based on binding of DNA primers with specific

targets in PCR amplicons can provide a rapid and

sensitive method of diagnosing drug resistance

This study utilizes the PCR-ELISA format and is

likely to be more economical.

EPIDEMIOLOGY

Discriminatory typing methods would have an

important place in the confirmation of clusters in

outbreak investigations especially in areas where

the disease is highly endemic. In the last 10

years,technique is restriction fragment length

polymorphism (RFLP) analysis of chromosomal

DNA using IS6110.

MOLECULAR DIAGNOSTICS FOR HOST

SUSCEPTIBILITY

The future of the molecular diagnostic test for

tuberculosis will no longer be confined to the

bacterial genome. The unfolding of the human

genome has led to the discovery of more

susceptibility or resistance genes associated with

tuberculosis.These genes are related to effective

killing of intracellular mycobacteria or granuloma

formation.

An extension of the principle of solid phase

detection of nucleic acids -- Using high-density

DNA probe arrays on a microchip, the system can

provide rapid strain identification, assessment of

drug resistance in cultured isolates, To determine

the host genetic markers that favour the

progression of any particular disease/infection

and Cellular responses to infection.

The use of microarray for a host genome survey

of tuberculosis susceptibility would not be too far

from reality.While these microarrays are

technologically advanced, the associated cost can

be great

Molecular based diagnostic methods wherever it

is practised at present in India should be used

only as an added adjunct in the diagnosis of

tuberculosis under special circumstances and

always in conjunction with the existing,

established diagnostic techniques i.e. sputum

microscopy and culture, and X-ray.

References

1.Molecular Diagnostics in Tuberculosis,Vincent Chichung ,Prepublished version—google 2.Molecular methods in the diagnosis of Tuberculosis,Indian J Tuberc2006:53:61-63 3.Diagnosis of m.tb using molecular biology technology,Juan Garberi :Asian Pacific Journal of Tropical Biomedicine,2011. 4.Recent Advances in rapid Laboratory Diagnosis of TB,Dr.WC YAM, Medical Bulletin, VolII, No1:Jan2006. 5.Advances in Molecular Diagnosis of TB,Dr.VM Katoch,MJAFI,vol59,No3;2003. 6.Topley and Wilson ,10th edition,Bacteriology. 7. Manual of Pre conference workshop on“ Update on Tuberculosis diagnostics & Therapeutics , NIZAMS,Hyderabad;2008

***

Bulletin of IAMM AP Chapter 7 Vol.17(1)

IDENTIFYING CLINICALLY IMPORTANT ANAEROBES- A DEMANDING TASK

Dr.K.Prasanthi, Associate Professor of Microbiology, Gandhi Medical College, Secunderabad

Dr.B.G.Viswanath, Final year Post Graduate, Gandhi Medical College, Secunderabad

Introduction : Anaerobic bacteria are organisms that are capable of surviving and growing in an atmosphere of little or no oxygen. Anaerobic bacteria can be further classified based on their relationship to oxygen, into obligate, aerotolerant or facultative anaerobe. They can be found in a variety of environments ranging from soil, water and humans, animals. Many anaerobic bacteria are found normally in the human body. In the intestines of humans anaerobic bacteria outnumber aerobic bacteria at a ratio of 1,000 to 1. Anaerobic bacteria have been encountered in infections involving virtually every organ and anatomic regions of the body. Within the past few decades, however anaerobic infections have become far more common either due to improved laboratory recovery of anaerobic bacteria or increased population of the patients, receiving immunosuppressive drugs for malignancy and other disorders resulting in compromising host resistance. The importance of anaerobes in certain infections is enhanced by the failure to provide appropriate antibiotic coverage for anaerobes in mixed aerobic – anaerobic infections and an increase in the number of anaerobes that have become resistant to antimicrobial agents. Misdiagnosed or over looked - It is essential to isolate anaerobes because infections are associated with high morbidity and mortality and the treatment varies with bacterial species involved. Most anaerobic infections involve a mixture of anaerobic and facultative anaerobe organisms, so that it is problematic to establish the extent to which a particular anaerobic species is contributing to infection. In addition, as ubiquitous members of our normal flora, anaerobic organisms frequently contaminate clinical materials. For these reasons, assigning clinical significance to anaerobic bacteria isolated in the laboratory is difficult.

Difficulties in the lab - Working only with pure cultures of organisms is a major principle of bacteriology, but anaerobic bacteria may be particularly difficult to isolate in pure culture, because indigenous anaerobes are often present in large numbers as normal flora on mucosal surfaces, even minimal contamination of a specimen can give misleading results. Right time – findings suggestive of anaerobic infection are – - Fetid or putrid odour from volatile short chain

fatty acids and amines is always associated with the presence of anaerobes in the sample

- Gas at the site of lesion - Location of infection in proximity to a mucosal

surface - Unique morphology on Gram stain - Failure of organisms seen on Gram stain of

original exudate to grow aerobically - Anaerobic growth on Selective media - Characteristic colonies on anaerobic agar plates

The lethal effect of atmospheric oxygen must be nullified until the specimen can be processed in the laboratory. Therefore all specimens must be injected into some type of oxygen free transport tube or vial. Right samples - include blood specimens, aspirates of body fluids (pleural, pericardial, cerebrospinal, peritoneal and synovial fluids), urine collected by percutaneous suprapubic bladder aspiration, abscess contents, deep aspirates of wounds, and specimens collected by special techniques, such as trans tracheal aspirates or direct lung puncture. Specimens of the lower respiratory tract are difficult to obtain without contamination. Double lumen catheter bronchial brushing and broncho alveolar lavage, cultured quantitatively, are useful. Direct-needle aspiration is probably the best method of obtaining a culture.

Bulletin of IAMM AP Chapter 8 Vol.17(1)

Specimens obtained from sites that are normally sterile may be collected after thorough skin decontamination. If aspirating a vesicle, fluid from the base of the lesion is collected. Avoid transport of aspirated material in the syringe. Tissue samples and biopsies are also very good specimens for anaerobic culture as anaerobes survive well in pieces of tissue. Punch biopsies, bone, or any other tissue specimen may be collected surgically. The specimen is placed in a sterile container without formalin. Avoidable specimens - Indigenous anaerobes are often present on the surfaces of skin and mucous membranes in large numbers, even minimal contamination of a specimen with normal flora can give misleading results, therefore have no diagnostic value. Use of swabs is always a poor alternative.

The following is a list of specimens that are likely to be contaminated with anaerobic normal flora and are not routinely accepted for anaerobic culture -

Throat or nasopharyngeal swabs

Gingival or other intraoral surface swabs

Expectorated sputum

Sputum obtained by nasotracheal or endotracheal suction

Bronchial washings or other specimens obtained via a bronchoscope

Voided or catheterized urine

Vaginal or cervical swabs

Gastric and small bowel contents (except for "blind loop" or bacterial overgrowth syndrome)

Feces (except for specific etiologic agents such as C. difficile and C. botulinum)

Rectal swabs, Surface swabs from ulcers and wounds (collect material from below the surface)

Any material adjacent to a mucous membrane that has not been adequately decontaminated

Right transport - Prompt delivery of specimens to the laboratory is essential. Various transport devices are available that generate an oxygen-free environment. These systems generally contain mixture of carbon dioxide, hydrogen and nitrogen, plus an indicator that illustrates aerobic conditions. After collection all air bubbles are expelled from the syringe and the specimen should be placed into an anaerobic transporter as soon as possible.Liquid

specimens are inoculated into a commercially available anaerobic transport vial. Swabs (the least desirable specimen) are placed in sterilized tubes containing carbon dioxide or prereduced, sterile Cary - Blair semisolid media. Two newer swab systems containing Amies medium and gel have been found to satisfactorily preserve viability of most anaerobes for at least 24 hours. Tissue pieces and curettings are transported in an anaerobic pouch which contain components to generate an anaerobic atmosphere, or in an anaerobic transport vial with a screw top Hungate cap. Examples of Transport media - Anaerobic transport media (ATM)- contains buffered mineral salts in a semi-solid media with sodium thioglycollate and cysteine added to provide a reduced environment. Resazurin or methylene blue is added as a redox indicator to reveal exposure to oxygen. This media provides an environment, which maintains viability of most microorganisms without significant multiplication and allows for dilution of inhibitors present in clinical material. Thioglycollate broth – Pancreatic digest of casein, Soy broth and glucose – enrich growth of most bacteria. Thioglycollate reduce the Eh. May be supplemented with hemin and Vitamin K1 – non selective for cultivation of anaerobes as well as facultative anaerobes. Thioglycollate broth serves as a backup source of culture material. Essential components in Anaerobic media - In all these media, dextrose serves as an energy source, peptones provide nitrogenous compounds, and yeast extract supplies B vitamins. Sodium chloride is incorporated to provide essential electrolytes. L cysteine, Sodium bisulfite, a reducing substance, is added to maintain reduced conditions and a low pH. Sheep blood allows the demonstration of hemolytic reactions. Hemin and vitamin K are growth factors. Vancomycin is employed to inhibit the growth of gram-positive organisms and Kanamycin or Gentamycin to inhibit aerobic, gram-negative facultative anaerobic bacilli. Right Processing – Microscopy - Direct Gram stain (methanol fixed and 0.1% basic fuchsin as the counterstain instead of safranin) from the sample is a must as it is an

Bulletin of IAMM AP Chapter 9 Vol.17(1)

important rapid tool which provides immediate information about the types of organisms present and presumptive evidence about the presence of anaerobic bacteria, to suggest appropriate initial therapy and serve as a quality control on the final culture analysis. Culture - Initial processing of anaerobic specimens involves inoculation of appropriate media. Primary plating media ideally should be prereduced or freshly prepared or used within 2 weeks of preparation. Plates stored for longer periods accumulate peroxides and become dehydrated, this results in growth inhibition. Reduction of media in an anaerobic environment eliminates dissolved oxygen. Prereduced, anaerobically sterilized (PRAS) media are commercially available and have an extended shelf life of up to 6 months. Examples of anaerobic media: Anaerobic blood agar -made of Columbia, Schaedier, CDC , Brucella, or BHI agar base supplemented with 5% sheep blood , 0.5% yeast extract, hemin, L cysteine and Vitamin K - Non selective for isolation of Anaerobes and facultative anaerobes . CDC Blood agar -is a tryptic soy agar base supplemented with Vitamin K1 and hemin. Bacteroides bile esculin agar (BBE) –Trypticase soy agar with esculin, ferric ammonium citrate and hemin, bile salts and gentamycin act as inhibitors - Selective and differential for Bacteroides fragilis group. BBE agar is also useful medium for Bilophila species, which usually produce black centered colonies due to H2S production. Laked Kanamycin Vancomycin blood agar (LKV) – Brucella agar with Kanamycin 75µg/ml, Vancomycin 7.5 µg/ml, Vitamin K1 10µg/ml, and 5% laked blood - selective for isolation of Prevotella and Bacteroides sps. Reduced Vancomycin concentration (2µg/ml) may be used when Porphyromonas species are looked for. Anaerobic phenyl ethyl alocohol agar ( PEA) - Nutrient agar , 5% blood, phenyl ethyl alcohol – selectively inhibits facultative GNB, prevents swarming by some Clostridia and Proteus. Egg yolk agar (EYA) – Egg yolk base – non selective for determination of lecithinase and lipase production.

CefoxitinCycloserine Egg Yolk agar (CCEY) – Egg yolk emulsion, lysed horse or sheep blood with Cefoxitin (8µg/ml) and Cycloserine (250µg/ml) selective supplement - selective for Clostridium difficile Cycloserinecefoxitin fructose agar (CCFA) - Fructose , Cycloserine 500mg/L, Cefoxitin 16mg/L, neutral red indicator - selective for Clostridium difficile . Cooked meat or chopped meat broth – solid meat particles initiate growth of bacteria, reducing substances lower oxidation reduction potential (Eh) - nonselective for cultivation of anaerobic organisms, with addition of glucose can be used for gas - liquid chromatography. Anaerobic Culture Techniques The techniques for cultivation should provide optimal anaerobic conditions throughout processing either by the anaerobic jars or boxes, plastic anaerobic bags, anaerobic chamber or glove box.The primary plating media for inoculating anaerobic specimens include one or more selective media and a nonselective anaerobic blood agar. The use of selective media along with nonselective one increases the recovery rate and can shorten the time to identification of organisms. Also for aerobic culture 5% sheep blood agar, Chocolate agar and Mac Conkey agar are set up because most anaerobic infections are polymicrobial and may include aerobic or facultative anaerobic bacteria. A back up broth, usually thioglycollate, to enrich for small number of anaerobes also be included.However, this media alone should never be used as a substitute for a solid media. Cultures should be placed immediately under anaerobic conditions and incubated for 48 hours or longer. The cultures should not be exposed to air until after 48 hours of incubation, since anaerobes are most sensitive to oxygen during their logarithmic phase of growth. Blood culture techniques:

Liquid media : A number of commercially available media appear to be satisfactory for recovery of anaerobic bacteria like tryptic soy broth, thiol broth and Columbia broth (BBL), trypticase soy broth (BD), thioglycollate medium, Septi – Check (Roche), Wilkins Chalgren broth ( Hemoline;

Bulletin of IAMM AP Chapter 10 Vol.17(1)

Biomerieux) and modified Schaedler broth (BIO ANAER).

Automated systems :Available are BACTEC (Becton – Dickinson) non radiometric systems and the BacT / Alert (OrganonTechnika Corp.).

Anaerobic Incubation methods : Jar Techniques:

Anaerobic jars: These were introduced in 1916 by McIntosh and Fildes. Introduction of cold catalysts and commercial packets that generate hydrogen and carbon dioxide after the addition of water greatly facilitated work in the clinical microbiology laboratory. Production of heat within a few minutes and subsequent development of moisture on the walls of the jar are indications that the catalyst and generator envelope are functioning properly. Reduced conditions are achieved in 1 to 2 hours, although the methylene blue or resazurin indicators take longer to decolorize. Anaerobic jars are made of polymetacarbonate,3.5L size, with pressure gauge, safety valve, Schrader inlet and outlet valves, low temperature catalyst and a carrier for 12X90mm petriplates.

BD Gas pak anaerobic system – contains an envelope that contains one tablet Sodium borohydride, one tablet Sodium bicarbonate plus citric acid. With the addition of water hydrogen is generated from Sodium borohydride which combines with oxygen in the jar in the presence of palladium catalyst to form water. Approximately 4 – 10% CO2 as measured by CO2 gas analyzer 1 hr after activation is generated from Sodium bicarbonate plus citric acid tablet. The CO2 is provided to stimulate the growth of anaerobes. 1 hr after activation the CO2 ranged from 4.6 to 6.5% and the oxygen concentration ranged from 0.2 to 0.7%.

The Anoxomat- (Mart BVR, Laboratorium, Holland) uses an automated evacuation replacement technique to create an anaerobic or Microaerophilic environment

in the jar. The anaerobic program repeats the evacuation replacement cycle 3 times. During each cycle the anaerobic gas mixture replaces 80% of the jar contents by drawing a vacuum of 25 inches (62.5 cm) of mercury. After 3rd cycle any remaining oxygen and any oxygen liberated from the media in plates is removed and final fill of the jar is made with the gas mixture containing 80% to 90% nitrogen, 5% to 10% hydrogen and 5% to 10% carbon dioxide, a reaction made possible by a palladium catalyst. This system also provides internal quality assurance programs that perform additional tests, when selected such as check for leaks, a test for catalyst activity, and a test for sufficient gas supply.

Anaerobic Bag Techniques: These are useful for laboratories processing small numbers of anaerobic specimens. Besides, the pouch can also be used for specimen transport. These permit visualization without disrupting the gaseous atmosphere.

Gas Pak EZ gas generating system – Brewer and Algeier in 1965 first introduced a hydrogen generating envelop. The GasPak EZ gas generating sachet consists of a reagent sachet containing an inorganic carbonate, activated carbon,ascorbic acid. When the sachet is removed from the outer wrapper, the sachet becomes activated by exposure to air. The activated reagent sachet and the specimens are placed in the resealable pouch and sealed. The sachet rapidly reduces the oxygen concentration within the pouch, at the same time inorganic carbonate produces carbon dioxide.

Anaeropack absorbs oxygen and generates carbon dioxide but does not generate hydrogen.

Anaerocult – catalyst free system makes use of wetted (rusting) iron fillings in a sachet to which water is added producing an oxygen free, carbon dioxide rich atmosphere.

Bio-Bag environmental type A - It is a transparent , individual , disposable

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environmental chamber that contain a gas generator consisting of one tablet of potassium borohydride and sodium bicarbonate and an ampule of hydrochloric acid, a catalyst cup containing palladium catalyst and an indicator containing an ampoule of resazurin which monitors the oxygen level. When an ampule of HCL is crushed the acid reacts with the tablet resulting in the formation of hydrogen and CO2. Hydrogen in the presence of palladium catalyst quickly reacts with the atmospheric oxygen in the bag to form water. Removal of oxygen is indicated by the change in colour, red to colorless, of the resazurin indicator included in the system.

Roll - Tube Technique: Developed by Hungate and popularized for clinical work by Holdeman and Moore, is useful for recovery of strict anaerobes when an anaerobic chamber is not available. These PRAS tubes can be inspected at any time without disturbing anaerobic conditions. The principal disadvantage is it requires more training time, cumbersome to isolate and purify strains and colonial morphology frequently not distinctive. Anaerobic chamber Techniques: Anaerobic chambersor glove boxes are made of molded or flexible clear plastic. Specimens and other materials are placed in the chamber through an air lock. The technologist uses gloves or sleeves that form air tight seals around the arms to handle items inside the chamber. Media stored in the chamber are kept oxygen free, and all work on a specimen is performed under anaerobic conditions. A gas mixture of 5% CO2, 10%H2, and 85% N2 and a palladium catalyst maintain the anaerobic environment inside the chamber. Anaerobic blood culture: Instrumented blood culture systems have special head space anaerobic atmospheres containing nitrogen and carbon dioxide without oxygen. BACTEC anaerobic blood cultures: Lytic / 10 anaerobic / F culture vials – (prereduced enriched Soybean casein digest broth with CO2) are used for anaerobic blood cultures with the BACTEC fluorescence series instruments for the qualitative culture and recovery of anaerobic microorganisms

from blood. Each vial contains a chemical sensor which can detect increases in CO2 produced by the growth of microorganisms. The sensor is monitored by the instrument every 10 minutes for an increase in fluorescence, which is proportional to the amount of CO2 present. A positive reading indicates the presumptive presence of viable organisms in the vial. BacT/Alert FAN bottles: contain BHI broth supplemented with Ecosorb that consists in part of Fuller’s earth and activated carbon particles. Ecosorb nonspecifically binds with the inhibitory factors present in blood and there by improves microbial recovery. Adequately filled bottle was defined as one containing 8 to 12 ml of blood. All bottles were logged into the BacT/Alert blood culture system and were placed in data units. Bottles flagged as positive were removed from the data units and an aliquot of blood broth mixture was removed aseptically with a needle and syringe. The aliquot was divided with one part being used for gram stain and the other being used for subcultures. Bottles with negative gram stains were returned to the data units for additional incubation and testing. Instrument negative bottles were left in the data units for 7 days, and no blind subcultures were indicated. Media and incubation of cultures: The combination of selective and differential agar plates yields information that suggests the presence and perhaps the types of anaerobes. All colony morphotypes from the non selective agar should be characterized and subcultured to purity plates and the backup broth should be gram stained. If cellular types are seen that were not present on the primary plates, the broth should be subcultured. The cultures should not be exposed to air until after 48 hours of incubation, since anaerobes are most sensitive to oxygen during their logarithmic phase of growth. Negative cultures are held for a minimum of 7 days before final examination. Thioglycollate broth should be inspected for turbidity accompanying a set of negative plates daily. When turbidity is seen, subculture a few drops onto a blood agar plate (anaerobic incubation) and onto a chocolate or blood agar plate for CO2 incubation.

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Examination of cultures, isolation of Anaerobes and Preliminary identification: Anaerobes are usually present in mixed cultures with other anaerobes and or aerobic or facultative bacteria. It is important to study all colony morphotypes, since facultative and anaerobic bacteria sometimes have very similar colony appearances. Clues to the presence of anaerobes in a culture include: (1) a foul odour upon opening an anaerobic jar or pouch, (2) more colony types present on the anaerobically incubated Blood agar plate than on the CO2 incubated plate, (3) growth on the selective media, and (4) red fluorescing or black pigmented colonies on LKVB or Blood agar. The primary anaerobic plates are examined and the quantity may be described as light, moderate or heavy or by the quadrants in which the growth occurs (1+, 2+, 3+, 4+ growth). All different colony types observed should be described in terms of size, shape, edge, profile, colour, opacity, pigment, hemolysis, pitting and distinct odour. Gram stain each different colony type and subculture onto a pair of Blood Agar plates for anaerobic and aerobic incubation, another set of Chocolate agar and Blood agar for CO2 incubation (to check aero tolerance), EYA for suspected Clostridum, Fusobacterium and Prevotella species for demonstration of lecithinase, lipase and proteolytic activities. Adopt special streaking methods by streaking the first and second quadrants of an anaerobic BA plate back and forth several times to ensure even lawn of heavy growth and streak the other quadrants for isolated colonies. Place the special potency disks ( Vancomycin 5µg, Kanamycin 1000µg, Colistin 10µg) on the first quadrant well apart from each other. These disks serve as an aid in grouping the anaerobic bacteria and for confirming the gram reaction, but do not imply susceptibility of an organism for antibiotic therapy. A 10µg Sodium polyethanolsulphonate (SPS) disk may be placed near the colistin disk for rapid identification of Peptostreptococcus anaerobius. If processing is done on open bench, incubate all anaerobic plates promptly within 20 min. Preliminary grouping of anaerobes and even complete identification of some organisms are based on colonial and cellular

morphology, Gram reaction, hemolysis, pigment production, susceptibility to the special potency disks (generally gram positive organisms are sensitive to Vancomycin and resistant to colistin , whereas the gram negative organisms are resistant to Vancomycin disk), catalase, indole and nitrate reactions. Special potency disc patterns : Bacteria Vanco-

mycin ( 5µg )

Kana- mycin (1000µg)

Colistin ( 10 µg )

Bacteroides fragilis group R R R

Bilophila / Sutturella species

R S S

Fusobacterium species R S S

Prevotella species R RS V

Porphyromonas species S R R

Veillonella species R S S

Gram positive anaerobic cocci

S V R

Gram negative R V V

S-sensitive, R-Resistant, RS–mostly resistant but can be sensitive Conventional methods for identification of anaerobes Classical Techniques for identifying anaerobes based on phenotypic characteristics will be presented by Diane Citronand Mike Cox. These methods will include appearance on gram-stain, growth characteristics and colony morphology on selective media, susceptibility to special potency antibiotic disks( proposed by Sutter and Finegold), preformed enzyme reactions, and classical biochemical reactions. Identification of Anaerobes - Kits containing biochemical tests, Rapid enzymatic tests are commercially available. These are good with fast growing anaerobes (i.e. Bacteroides fragilis and Clostridium perfringens). Most clinical microbiology laboratories are able to identify the major anaerobic bacteria. Peptostreptococci are generally not speciated because they are generally susceptible to commonly used antimicrobials. Clostridium spp. can be identified by the presence of spores and their ability to survive 30 minutes exposure to ethanol or heating to 80°C for 10 minutes. Nonspore forming Gram positive bacilli can be speciated by gas liquid

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chromatography and biochemical tests. Propionibacterium spp., which is often a contaminant, can be separated from other nonspore forming Gram positive bacilli by a catalase test and indole reaction. B. fragilis group grows on 20% bile and is generally catalase positive. Common pitfalls in specimen processing and isolation procedures: 1) Failure to prepare the Gram stain directly from the clinical specimen which alerts on to the possible presence of organisms requiring special media or conditions of incubation. 2) Use of thioglycollate or other liquid medium as the only system for growing anaerobes, as a number of anaerobes will not grow in thioglycollate medium. 3) Failure to check liquid medium if growth is recovered only from anaerobic plates. A Gram stain of the broth often alerts one to the presence of additional anaerobes. 4) Use of inadequate media. Failure to use fresh media. 5) Failure to do quality control on media and reagents for performance. 6) Failure to use supplements in media like Vit K1 and hemin. 7) Failure to use selective media. Some anaerobes may be overgrown by more rapidly growing organisms and overlooked if selective media are not used. 8) Failure to hold cultures for extended periods as some fastidious organisms may require 5 – 7 days to grow. 9) Failure to minimize exposure of colonies on plates to air. For some fastidious anaerobes, > 15 min exposure can cause loss of viability.

Automation in identification of Anaerobes : Miniaturized biochemical systems : OmniLog ID : ( Biolog microbial ID system) This System fully automates the process of microbial identification by incubating, reading and interpreting results from up to 50 BiologMicroPlates™ at a time. Biolog AN microplate is designed for identification of a very wide range of anaerobic bacteria. It is based on the reduction of tetrazolium , responds to the process of metabolism (oxidation of substrates) rather than to metabolic by products. Works with any carbon source and no color developing chemicals need to be added after incubation. The anaerobe data base contain over 350 taxa of anaerobic bacteria including over 70 species of lactic acid bacteria.

Performs 95 discrete tests simultaneously and gives a characteristic reaction pattern called a metabolic fingerprint. The patterns are compared using Biolog data base software to give an identification. BBL brand crystal Anaerobe ID - Becton Dickinson contain 29 dried biochemical and enzymatic substrates . A bacterial suspension in the inoculums fluid is used for the rehydration of the substrates. The tests used in the system are based on microbial utilization and degradation of specific substrates detected by various indicator systems . Enzymatic hydrolysis of fluorogenic substrates containing coumarin derivatives of 4methylumbelliferone (4MU) or 7 amino – 4 methyl coumarin(7AMC), results in increased fluorescence that is easily detected visually with a UV source. Chromogenic substrates upon hydrolysis produce color changes that can be detected visually . RapID ANA II - Remel : Four hour identification of over 90 medically important anaerobes based on enzyme technology. Reagent impregnated wells in clear plastic tray are provided with kit. API 20A, Biomerieux, OxoidMicrobact, API- ZYM BIOMERIEUX, Rapid ID 32 A BIOMERIEUX Minitek, Crystal Anaerobe identification system (BD) Rapid Anaerobe identification panel : Vitek ANC card, Biomerieux : BioMerieux (Marcy, France) has developed a new colorimetric identification card (ANCcard) which in conjuction with the Vitek 2 system , permits this automated and widely distributed identification system to identify 63 taxa, including 49 taxa of anaerobic bacteria. The ANC card is based on established biochemical methods and newly developed substrates. There are 36 biochemical tests measuring carbon source utilization and enzymatic activities. Final results are available in approximately 6 hours. Using a sterile stick or swab prepare a homogenous suspension with a density equivalent to a McFarland No. 2.70 to 3 .30 using a calibrated vitek DENSICHEK. The offline tests selected for use in the ANC product are Gram stain , morphology, and aerotolerence. As part of the ID process, the software compares the test set of reactions to the expected set of reactions of each organism, or organism group, that can be identified

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by the product. A quantitative value , the percent probability , is calculated and relates to how well the observed reactions compare to the typical reactions of each organism. A perfect match between the test reaction pattern and the unique reaction pattern of a single organism , or organism group, would provide a percent probability of 99. When a perfect match is not obtained , it is still possible for the reaction pattern to be sufficiently close to that of an expected reaction pattern such that a clear decision can be provided about the organism identification. The range of percent probabilities in the one choice case is 85 to 99. Values closer to 99 indicate a closer match to the typical pattern for the given organism. Microscan Rapid anaerobe panel ( Dede Behring) Determination of metabolic products by GLC: Analysis of metabolic products by GLC is a practical , inexpensive procedure which improves the speed and accuracy of identification. Metabolic products released in to the broth culture media during anaerobic growth are key characteristics of obligate anaerobic bacteria and many facultative anaerobic bacteria. Metabolic pathways represent genetically stable or conserved traits of bacteria. It is used to separate and identify anaerobic metabolic end products ( volatile fatty acids and non volatile organic acids) of carbohydrate fermentation and amino acid degradation. PYG broth can be used for analysis of acid metabolic products. Use of GLC to detect anaerobes in exudates and body fluids has been described .However direct GLC provides only presumptive clues , and should be interpreted cautiously in polymicrobial infections. Identification of volatile fattyacids : GLC is used for identifying short chain volatile fatty acids that are soluble in ether include acetic, propionic , isobutyric, butyric, isovaleric,valeric, isocaproic, caproic. These are identified by comparing elution times of products in extracts with those of a known acid mixture chromatographed under the same conditions on the same day. Analysis of Nonvolatile acids : Pyruvate, lactic, Fumaric, succinic, hydrocinnamic and phenyl acetic acids are not detected with the ether extraction procedure for volatile fatty acids. These nonvolatile acids are identified after preparation of methylated derivatives. Identify

nonvolatile or methylated acids by comparing elution times of products in extracts with those of known acids chromatographed on the same day. High resolution gas liquid chromatography: Expanded for the analysis of cellular fatty acid analysis (longer chain fatty acids) to produce chromatograms for identifying organisms often without the need for other phenotypic information. One such commercial system MIDI microbial identification system has more than 600 bacteria in the chromatographic database. MIDI instant Anaerobe for Sherlock : New procedure takes 2 – 3 minutes to prepare an extract from 2.5mg of pure culture. Once the extract is prepared only 9 minutes are required to identify the isolate. The sherock system identifies microbes by analyzing the fatty acids present in the cell membrane . The fatty acids are separated from the cell and then analyzed with an Agilent technologies gas chromatograph (GC). The Sherlock pattern recognition software then compares the fatty acid pattern of each sample to libraries of stored patterns to make an identification. Identification process takes less than 15 minutes from pure cuture compared to 24 hours required of most anaerobe identification.Uses 2.5mg of cells , compared to 20mg of cells required for other ID systems. No biochemical tests, gram stain or biochemical cards are required. 3 reagents and only 250 µl of each required for sample preparation. Immunological tests: The direct detection of anaerobic antigens had limited successful applications. Fluorescent antibody kits developed commercially are with nonspecific and insufficient sensitivity to aid in direct detection of organism in fresh samples. Immunological procedures for the direct detection of C.difficile toxins in fecal samples include several commercially available EIAs. Other tests for C.difficile include latex agglutination test and an immunoassay for the glutamate dehydrogenase common antigen and gas liquid chromatography to detect isocaproic acid. Specificity has been an issue for these methods. Toxin A is enterotoxin and toxin B is cytotoxic. Both toxins are detected by the C. DIFFICILE TOX A/B II™.

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Membrane EIA single test for the qualitative detection of Clostridium difficile Toxins A and B in Stool Specimens Molecular genetic methods: Nucleic acid probes are not yet standardized and are not commercially available for clinically important anaerobes. Probes are most useful for rapid identification of colonies directly from primary plates or pure cultures. Methods such as 16S rRNA PCR-RFLP profile determination, can help to distinguish species of anaerobes that are otherwise difficult to differentiate. Molecular methods have demonstrated the spread of specific resistance genes among the members of the Bacteroides genus. A search for toxin genes and ribotyping may promote a better understanding of the pathogenic features of some anaerobic infections, such as the nosocomial diarrhoea caused by C. difficile and its spread in the hospital environment and the community. Mass Spectrometry Years ago, labs used gas liquid chromatography to detect fatty acids that anaerobes produce when they grow. Patterns noted in tandem with identification systems and phenotype enabled identification. Recently, mass spectrometry "has resurrected itself" in the anaerobic identification realm, Dr. Hall says, as techniques such as matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) are used to detect proteins. Other Detection Methods Pyrosequencing - Sequencing is another new identification technique. Looking at the DNA makeup of organism about 300-500 base pairs for sequencing, and about 30 base pairs for pyrosequencing labs can build databases to make matches. Pyrosequencing is a type of DNA sequencing based on the “sequencing by synthesis” method. Pyrosequencing technology uses an enzyme cascade system, consisting of four enzymes and specific substrates to produce light upon nucleotide incorporation during DNA synthesis. The amount of light produced is proportional to the number of incorporated nucleotides. When light signal is detected, the base is registered and the next nucleotide is added. If the added nucleotide is

not complementary to the next base in the template, no light will be generated. Still in research, oligonucleotide arrays or microarrays based on the 16-23S rRNA intergenic spacer sequences to identify 28 anaerobic bacteria species and Veillonella have demonstrated 99.7% sensitivity and 97.1% specificity. Identification of an anaerobe to a species level is often cumbersome, expensive and time-consuming, taking up to 72 hours. Identification is most helpful in selecting an antibiotic against a species that has predictable antibiotic susceptibility. Occasionally, species identification of an organism will provide the diagnosis, as with Clostridium difficile in colitis or Clostridium botulinum in botulism. Identifying B. fragilis group, which often cause bacteraemia and septic complications, has significant prognostic value. However, because most anaerobes are endogenous, there are rarely epidemiologic reasons to perform complete identification.

ANTIMICROBIAL SUSCEPTIBILITY TESTING FOR ANAEROBES The antimicrobial susceptibility of anaerobes has become less predictable. Resistance to several antimicrobials, especially by anaerobic Gram negative bacilli (AGNB) and Fusobacterium spp., has increased. It is important to test susceptibility of anaerobes recovered from sterile body sites, those with particular epidemiologic or prognostic significance (e.g. C. difficile), or those that are clinically important and have variable susceptibilities. As disk diffusion test results for anaerobes do not correlate with those of the reference method, standard susceptibility testing methods have been established for testing anaerobic bacteria against various antimicrobial agents by CLSI. They are agar dilution method and broth microdilution method. But the fastidious nature of many species and the labor intensity of using these methods, many clinical microbiology laboratories perform these tests under special circumstances.Wilkins & Chalgren agar isrecommended forsusceptibility testing of anaerobes. (CLSI: Methods for

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antimicrobial testing of anaerobic bacteria, M11-A6, Wayne,Pa,CLSI.) Commercial method of E test and spiral Biotech may facilitate anaerobic susceptibility testing. Screening of AGNB isolates (particularly Prevotella, Bacteroides and Fusobacterium spp.) for beta-lactamase activity( Nitrocefin disks – Cefinase ; BBL) is helpful. However, occasional resistance is through other mechanisms is reported. Routine susceptibility testing is extremely time consuming and often unnecessary. It should be limited to isolates from blood, bone, central nervous system and serious infections, as well as to those recovered from any normally sterile site in pure culture and infections not responsive to empiric therapy. Antibiotics tested should include penicillin, a broad spectrum penicillin, a penicillin plus a betalactamase inhibitor, clindamycin, chloramphenicol, a second generation cephalosporin (e.g. cefoxitin), metronidazole, a quinolone (Levofloxacin), a macrolide (Erythromycin, Azithromycin) and a Carbapenem (Imipenem, Meropenem) . The method recommended by the Clinical laboratory standards institute (CLSI) includes agar dilution testing, microbroth and macrobroth dilution (inoculum adjusted to turbidity of a 0.5 McFarland). Newer methods include the E test and the spiral gradient end point system. Characteristics of Anaerobes with Medical importance Bacteroides species -are non-spore forming gram-negative bacilli and produce colonies which are typically > 1mm in diameter, circular, entire, convex and usually surrounded by a dark grey zone on BBE agar. Bacteroides spp.are considered one of the most important anaerobic bacteria because of their involvement in many infectious processes and their resistance to antibiotics. Bacteroides are the most common species of anaerobic bacteria isolated from soft tissue infections and bacteremia. The bacteria produce numerous virulence factors such as capsules, endotoxins and various enzymes that cause tissue destruction and have abscessogenic potential. Bacteroides species are among the most prevalent bacteria found in human feces. Prevotella spp. and Porphyromonas spp– are Gram negative coccobacilli and Gram negative bacilli respectively and produce

black / brown pigmented or red fluorescing colonies. To detect pigment, it is often helpful to pick up several colonies with a swab and look at the cell mass on the swab. The pigment of Prevotella is an iron protoporphyrin which protects the organism from oxygen toxicity and possesses inherent catalase activity. The capsular polysaccharide, inhibits opsono phagocytosis, promotes abscess formation and adherence to epithelial cells. Fusobacterium species - produce Bread crumb like or speckled, smooth, translucent, butyrous colonies that are subsequently shown to contain slender fusiform gram negative bacilli. They produce a characteristic rancid butter odour due to butyric acid production. Fusobacterium produce a biologically active endotoxin. Propionibacterium Species The Propionibacterium species are pleomorphic gram-positive bacilli that may appear oval, rod-shaped or club-shaped. Some may appear branching or forked also. This is a group of aerotolerant anaerobic bacteria that can grow and multiply in the presence of atmospheric oxygen. These anaerobic bacteria are normally found on the skin and have been associated with an inflammatory process in acne (Propionibacterium acnes). They can also cause endocarditis and bacteremia but are usually considered a contaminant and a non-pathogen. Actinomyces Species The Actinomyces species are straight to slightly curved gram-positive bacilli that can range from short rods to long, branching rods and form molar tooth shaped colony. This anaerobic bacterium is found normally in the oral cavity, upper respiratory tract and, to a lesser extent, the gastrointestinal tract. They are not a particularly virulent species of bacteria and usually only cause infection when there is some disruption in the surface of the oral cavity or upper respiratory tract (opportunistic infection). C. difficile Clostridium difficile produces a toxin leading to disease. Causes the most serious cause of antibiotic-associated diarrhea, often leading to pseudomembranous colitis, a severe colon infection that can in rare instances be fatal.

References: 1) Wadsworth – KTL Anaerobic Bacteriology Manual,

6th

ed. 2) Mandell, Douglas and Bennett’s Principles and

Practice of infectious Diseases, 7th

edition. 3) Koneman’s Color Atlas and Textbook of Diagnostic

Microbiology, 6th

edition. 4) Bailey & Scott’s Diagnostic Microbiology, 12

th

edition.

5) CLSI: Methods for antimicrobial testing of anaerobic bacteria, M11-A6.

***

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FLUORESCENCE MICROSCOPY

murtyds@gmail.com http://murtyds.blogspot.com/

Dr. D.S.Murty Assistant Professor of Microbiology

Osmania Medical College, Hyderabad

Introduction to Fluorescence

Fluorescence is a term used to signify a

compound’s ability to absorb light at one (shorter) wavelength and emit light of a longer wavelength. Generation of luminescence through excitation of a molecule by ultraviolet or visible light photons is termed as ‘Photoluminescence’. This is formally divided into two categories-Fluorescence and Phosphorescence. ‘Fluorescence’ describes light emission that continues only during the absorption of the excitation light. The time interval between the absorption of excitation light and emission of reradiated light in Fluorescence is extremely of short duration, usually less than a millisecond. If the emission of light persists for up to a few seconds after the excitation energy(light) is discontinued, the phenomenon is known as ‘Phosphorescence’.

Fluorophores

A variety of substances, either organic or

inorganic, will exhibit fluorescence in their natural form. This is termed as ‘primary’ or ‘autofluorescence’. But it is often faint and nonspecific. Later, many dyes have developed that have very bright fluorescence, excited by a specific wavelength of light and emit light of defined wavelength. These dyes are called, ‘Fluorophores’ or ‘Fluorochromes’. These are used to selectively stain parts of a specimen. This method is called, ‘secondary fluorescence’. These fluorochromes may be conjugated with other organic substances like antibodies and then are called, ‘Fluorescent probes’, which have wide application in ‘immunofluorescence’. Albert H Coons along with Nathan Kaplan has developed this Fluorescent antibody technique in 1941.

Examples of Fluorochromes:

Fluorochrome Excitation wavelength (nm)

Emission wavelength (nm)

Comment / staining

Acridine Orange 490 530, 640 590

Nucleic acids Acid organelle

Auromine 450-490 515 Mycolic acids

Calcofluor white 355-425 460 Cellulose (fungal cell walls)

Fluorescein isothiocyanate (FITC)

490 520 Antibody labelling

Texas Red 596 620 Antibody labelling

Acridine Yellow 470 550 Nucleic acids, acid fast cells

Rhodamine B isothiocyanate (RITC)

570 595 Antibody labelling

Acriflavin 430 515 DNA

Fluorescence Microscopy

The Fluorescence Microscope (FM) was designed in early part of 20th century (1911) by August Kohler, Carl Reichert and Heinrich Lehmann. However, the potential of this instrument was not realized for several decades. But, now it became an indispensable tool of Cellular biology with growing number of applications in Microbiology as well. Initially, the fluorescence microscopes were used to detect ‘autofluorescence’ of various biological specimens. In 1930s, Austrian Investigator Max Haitinger developed the technique of ‘secondary fluorescence’, which employs fluorochromes to stain specific tissue components and bacteria.

The basic task of FM is to irradiate the specimen with a desired and specific wave length of light and then to separate the much weaker and longer wave length fluorescent light emitted from the specimen. A FM also produces a magnified image of the sample, but the image is based on the ‘second light source’- the light emanating from the fluorescent specimen rather than the light originally used to illuminate(excite) the sample.

In order to see the fluorescent image with adequate contrast, the FM must be equipped with “Optical filters”, that transmit the required wave length of light. The ‘Excitation filter’ (also called ‘Primary filter’ or ‘exciter’), transmits the wavelength of light that excites a particular fluorophore, while the ‘barrier filter’ (or the ‘emission filter’) attenuates all the light transmitted by the excitation filter and efficiently transmits the fluorescent light emitted from the sample. These two types of filters are based on the excitation and emission peaks of the particular fluorophores used (light pathways will be discussed later under ‘epifluorescence’).

The earliest configurations used transmitted light for FM., and are referred to as “Transmitted light fluorescence microscopes”. In these, a short wave length light passes

through the excitation filter. Its transmission matches the excitation wave length of the fluorophore being used. This light reaches the specimen from below, through a dark field condenser. The fluorescence emanated from the specimen passes through a ‘barrier filter’ before reaching the eye. But, the specimen view in these early configurations was often very low in contrast and super imposed on a back-ground flooded with the scattered illumination, resulting in poor resolution and low brightness.

The most important advancement in FM was the development of ‘incident light’ (episcopic) illumination in 1929. An important element in these epifluorescence microscopes is a ‘beam splitter’, which ‘reflects the excitation light and transmits the emission light’. Initial instruments used half silvered mirrors as beam splitters. But, the most valuable contribution was the introduction of “dichromatic beam splitters” or “ Dichroic Mirrors” by Russian Scientist, Eugeny Brumberg in 1948. These Dichroic mirrors were commercialized by Ploem in 1967, and came into wide use in fluorescence microscopy. EPI-FLUORESCENCE MICROSCOPY:

Reflected light FM is the current method of choice. This is also called, “Incident light FM”, “Episcopic fluorescence” or simply, “ Epifluorescence”. The essential elements of an Epifluorescence microscope are : Light source, Filter set and Objective. I.) Light source: To generate excitation light of sufficient intensity, a powerful, compact light source is essential. The most common light sources are ‘Mercury arc lamps’ and ‘Xenon arc lamps’. These are expensive, require stable electric supply and have limited life ( of usually 200-300 light hours). These may explode when used beyond their life span. They also need a warm up time of about 20 minutes , and if switched off, they must cool before switched on

again. The ‘quartz halogen’ lamps are increasingly gaining popularity over the arc lamps in most applications. They usually have a longer life span (up to 2000 light hours). In normal light microscopy, 6 W- 12W lamps are used, but 100-200 W lamps are required for FM . In the past few years, Laser sources like ‘argon’ or ‘argon-krypton’ lasers are being chosen in research settings because of their high luminescence, small source size and low divergence. Lasers are used in ‘Confocal microscopy’ to produce a sharp image. II.) Filter set : The epifluorescence microscopes require an efficient filter system that can reduce the observed excitation light to the minimum and transmit almost all of the available fluorescence signal. This increases “ Signal-to-Noise” ratio. The primary filtering element in EpiFM is a set of three filters housed in the “FILTER CUBE”. 1. The “excitation filter” : transmits the wave

length of light, that efficiently excites a particular fluorophore. Common filter blocks are named after the type of excitation filter. Examples:-

a. “UV” or “U” for Ultraviolet excitation light (<400 nm) ; suitable for ‘Hoechst 33258’, a dye used in Mycoplasma detection.

b. “B” for Blue excitation light (450-500 nm) , suitable for FITC, Acridine orange, Auramine-Rhodamine etc..

c. “G” for Green excitation light(500 -570 nm), for Texas Red etc.

2. The “emission filter” (Barrier filter) : blocks

the shorter wave length of light (transmitted by excitation filter) and transmits any fluorescence (longer wave length) light emitted from the specimen.

3. The “Dichroic Mirror” (Dichromatic beam

splitter): is an unique component of the epifluorescence systems. It is a thin piece of coated glass, set at an angle of 450 to the light path. It reflects > 90% of the shorter

wave length(excitation) light and transmits > 90% of the longer wave length(emission) light.

III.) Objective : In all forms or ‘reflected’ light microscopy, the image intensity is a function of the numerical aperture(NA) of the objective and magnification(M).

Image intensity (μ ) = NA 4 / M 2 From this equation, it can be observed that , the brightest images can be gathered by the objective of high NA and low magnification. For example, an objective of 40x or 60x will produce brighter images than 100x objective. In general, high NA (1.3-1.4) oil immersion plan fluorite or plan apochromatic objectives produce brightest fluorescent image. Many newer forms of Epifluorescence Microscopes are provided with a means to change these optical elements in a convenient manner by arranging a set of four of more filter cubes in a circular or linear ‘turret’. A specific filter cube can be selected depending on the fluorophore being used, in a manner similar to that of selecting a required objective in optical microscopy.

Working with a fluorescence microscope

In order to optimize the functioning of a FM to visualize a bright or even a faint image against a dark (or faint) background, the image brightness and resolution must be maximized applying the optical principles that have been discussed so far.

The first rule in fluorescence microscopy (and all other forms of microscopy, in fact) is to keep the optical elements completely free of dust, dirt, oil, solvents, and any other contaminants. The microscope should be kept in a low vibration flat surface in a smoke-free room that is as clean as possible and has minimal disturbance of the circulated air. A dust cover has to be covered on the microscope when not in use and all accessories should be kept in air-tight containers. Avoid using corrosive solvents to clean any part of the microscope, and use only diluted soapy water to clean non-optical surfaces. Never drag anything across the lens surface with a high degree of pressure, including lens paper, to avoid the possibility of introducing very fine scratches onto the surface.

When using the oil immersion objective,

it is very much essential to blot the front lens with the tissue to remove oil, after examining each specimen, especially a positive specimen. It is also better to use an oil of low autofluorescence. The oil has to be applied to the objective-cover slip interface without formation of air bubbles. When cover slips have to be used with the ‘dry objectives’ with high Numerical aperture, the thickness of cover slips must be around 0.17 mm, as recommended by the ‘Royal Microscopical Society’. And oil should never be used with these dry objectives, as cleaning a dry objective can be much more difficult .

It is most critical to select the proper ‘Filter Cube’ appropriate for the fluorochrome being used. All the optical components must be in proper alignment to give good results. Objectives may be provided with iris diaphragm,

to adjust the illumination and to minimize the glare.

The microscope must be connected to a stable power supply of correct voltage, without any loose connections. The power cord must be periodically examined for any wear and tear. The microscope MUST be SWITCHED OFF and UNPLUGGED :- at the end of work; when there is a storm with lightening; when cleaning and when changing the lamp or replacing a fuse.

A ‘time log’ has to be maintained to

record the working time at each use, as the lamp needs replacement after working for limited number of hours, as specified by the manufacturer. If the ‘arc’ lamps are used beyond their life time, there is a risk of explosion. If the lasers are being used as a light source, it is necessary to follow all safety precautions recommended by the supplier as there is a risk of thermal injury to eyes if they are used improperly.

In ideal cases, all microscopy should be done in darkened rooms (for microscopes with built-in-illumination) , but this is especially important with fluorescence microscopy. Human eyes can not quickly adjust to darkness, so it will be difficult to see the very dim fluorescent specimen immediately after darkening the room. Hence, it is better to start working (focusing) after the eyes were adapted to the darkness.

It is also necessary to avoid excessive

bleaching of the specimen. This can be done by blocking the excitation light when not viewing or photographing the specimen. Autofluorescence can be minimized by thoroughly washing the specimen to remove excess fluorochrome prior to mounting. Modifications of the Fluorescent Microscopy :

With the development of the computerized control of the instrument and digital imaging and processing software, there were continued efforts to enhance the detection

of the emitted light, however weak it may be and to reduce the ‘back ground noise’. Various latest versions are :

1. Laser Scanning Confocal Microscopy 2. TIRFM (Total Internal reflection FM)

Laser Scanning Confocal Microscopy (LSCM)

In this, a coherent beam of laser is used

as a light source. It passes through a ‘pin-hole aperture’ that is placed in a “conjugate plane” (confocal) with a point in the specimen and a second pin-hole aperture situated in front of the detector ( a PMT- photomultiplier tube). The laser is scanned across the sample by a ‘galvanometer mirror scanner’ (in x-y plane). All the “out-of focus” emission is blocked by the pin-hole arrangement to reduce blurring. Refocusing the objective will shift the focal point in the specimen in to a new plane, to produce thin optical sections of the sample. All these images are re-constructed to give a 3-D image. This facilitates ‘z-axis scanning’ of thick specimens, which would appear blurred by conventional FM. Major application of LSCM involves imaging either fixed or living cells and tissues that have been labeled with one or more fluorescent probes.

Total internal reflection Fluorescence Microscope (TIRFM)

The light rays will be totally internally

reflected at the interface between two media with different refractive indices, when the ‘incident angle’ exceeds a ‘critical angle’. This phenomenon is exploited in TIRFM. This form of microscopy is often combined with “inverted microscope”.

The excitation light is made to pass at a higher incident angle through the glass cover slip or tissue culture plates, where the cells adhere ( Specimen). The light is totally reflected instead of refracting. But, in doing so, it induces an electromagnetic wave called, ‘evanescent wave’ in the specimen (stained with fluorophores). This generates a very thin electromagnetic field

(< 200 nm; much lesser than 600nm thin section that can be achieved in LSCM), and selectively excite the fluorophores in that restricted region only, which enhances image quality. This TIRFM will be a valuable tool in studying the cell- to-cell interactions. Live-cell imaging represents a most promising application of TIRFM technique.

Flow cytometry (FACS : Fluorescence activated cell sorting)

This is another instrument that uses the

fluorescence principle. This is useful to analyze the cells according to their fluorescence and light scattering properties. Cells in a mixed population are stained with monoclonal antibodies that are labeled with fluorochromes of different colors. Cells are kept in a sample chamber, from where they are expelled from a small vibrating nozzle, so that the cells are lined up in a single file. The cell stream passes through a laser beam. Photocells measure the fluorescence and light scatter, giving an index of cell size and granularity. A computer analyzes this data. As it leaves the nozzle, each particle(cell) receives some electric charge, based on the data from the computer. Then these cells pass through a pair of charged plates, where they deflected into different paths and get collected in collecting chambers. Applications of Flow cytometry :

1. Immunophenotyping : determination of profile of selected surface markers displayed by Leukemia cells and Leukemia typing;

2. CD4 count 3. Use of multiple monoclonal antibodies

with different colors to analyze different populations of cells to get information like -

How many cells express the target antigen

Percentage of cells with a particular marker

Antigen density in cells

Approximate size of the cells.

Preservation of Fungi Dr. D.Sudha Madhuri,

Assistant Professor of Microbiology, Gandhi Medical College, Secunderabad

Fungi are a diverse group of heterotrophic organisms that exists as saprobes, commensals or parasites.most of them are found on decaying vegetative matter and in soil. As fungi are such a diverse group, several methods of cultivation and preservation are required to ensure the viability and morphological, physiological, and genetic integrity of the cultures over time. The cost and convenience of each method however, also must be considered. Effective preservation requires cultures in viable state and should be free of contamination. The cultures must be restored to same condition prior to preservation and should not develop phenotypic and genotypic changes . The primary methods of culture preservation are : Continuous growth methods-the cultures are grown on agar, typically are used for short-term storage. Such cultures are stored at temperatures of from 5°-20°c, or they may be frozen to increase the interval between subcultures. The methods are simple and inexpensive because specialized equipment is not required. The maximum duration of storage with the species being preserved is 6 months. Drying- drying is the most useful method of preservation for cultures that produce spores or other resting structures. Silica gel, glass beads, and soil are substrata commonly used in drying. Fungi have been stored successfully on silica gel for up to 11 years (smith and onions 1983).drying methods are technically simple and also do not require expensive equipment freezing-freezing methods include cryopreservation, liquid nitrogen or in standard home freezers. The method is highly successful with cultures that produce mitospores. Freeze-drying and freezing below -135°c are excellent methods for permanent

preservation and highly recommend. These methods require specialized and expensive equipment.

Long term preservation methods verses short term preservation methods : Short term preservation methods

Continuous growth methods by Serial transfer of cultures and storing at temperature of 5-20 oC, and maintained up to 1 year.

Advantages of short term preservation

Simplest,Cryoprotectant not needed ,No need of expensive instruments.

Disadvantages of short term preservation

Time consuming and labour intensive

Not suitable for long term preservation, subcultures to be done 2-3 monthly

Many fungi tend to degenerate and may lose the ability to grow and fungi tend to lose ability to sporulate.

Chances of Contamination during storage and while transferring and Fungal cultures get infested by mites

Long-term preservation methods.

1. Oil overlay 2. Immersion in distilled water 3. Soil or sand 4. Freezing 6. Lyophilization 7 .Liquid nitrogen

1.Oil overlay : Low cost & low maintenance method. Fungal cultures on agar slant are covered with oil for 10 mm length. Entire surface should be submerged in oil. The tubes are kept in upright position at RT. The fungi are preserved for several years at RT or 15-20 oC. This method is appropriate for mycelial or nonsporulating fungi and Oil Reduces mite infestation. Oil level should be checked periodically.

Bulletin of IAMM AP Chapter 23 Vol.17(1)

Major disadvantage is that fungi continue to grow & selection of mutants that can grow under adverse conditions may occur. Another problem is How to retrieve a culture from mineral oil. 2. Immersion in distilled water Inoculate the fungal strains on preferred media & then incubate for several weeks at 25 oC to induce sporulation. Sterile DW (6-7 ml) is added aseptically to the culture and transfer it to cryovials containing DW or cut the culture in small pieces from agar slant by straight loop & transfer it to cryovials containing DW. Tubes should be completely sealed. Many fungi do survive for up to 7 yrs. It is Simple, Inexpensive & yet effective method. In CDC, Atlanta, USA 93% of fungi survived for 60 months. Water suppresses morphological changes. The fungus may be re-grown by dipping a sterile applicator stick into the water suspension, thereby retrieving some fungal cells, and aseptically inoculating them into a desired culture medium. After appropriate incubation, the inoculated medium should contain fungal growth resembling the original stored culture; pleomorphism is rarely seen in cultures retrieved from water storage. It is important to occasionally inspect the tubes and to add sterile water to any showing water loss. This technique is highly successful. Many fungi (including filamentous molds, yeasts and yeast-like fungi) will survive for a decade or more in sterile water. Some zygomycetous fungi may be less stable in water storage, surviving for only a few months, but most fungi commonly encountered in clinical laboratories or in plant pathology laboratories remain viable for years. 3. Soil or sand Some fungi: easily preserved & successfully maintained for many years in dry sterile soil or sand Dormancy caused by dryness can take time to develop. Glass bottles (60 ml) are filled to two-thirds capacity with sand or loam soil (water content 20%) then sterilized by autoclaving for 20 min at 120 oC. The bottles are allowed to cool and then sterilized again. Sterile DW is added to a culture, and then colony surface is scraped gently to produce 5 ml of spore or mycelial suspension. 1 ml of the suspension added to each bottle of soil or sand. After 12-14 days of growth at RT, the bottles are stored in the

refrigerator at 4°C. To retrieve the fungus, a few grains of soil are sprinkled onto fresh agar medium 4. Freezing Most fungal cultures frozen at -20° to -80°C in mechanical freezers remain viable for up to 5 years or more. Cultures grown on agar slants in bottles or test tubes with screw caps can be placed directly in the freezer Cryoprotectants -Because living cells can be damaged severely by freezing and thawing, chemical cryoprotectants are used in most protocols Cryoprotectants are of two types: Penetrating agents such as glycerol and dimethyl sulfoxide (DMSO), which readily pass through the cell membrane and protect intracellularly and extracellularly Nonpenetrating agents such as sucrose, lactose, glucose, mannitol, sorbitol, dextran, polyvinyl-pyrrolidone, and hydroxyethyl starch, which exert their protective effect external to the cell membrane Inoculate the fungal strain on preferred media & incubate up to resting phase. Cut the culture in small pieces about 4 mm from agar slant by straight loop & transfer to the cryovial containing 10% glycerol or 5% DMSO .Tighten the cap properly & arrange in the box & keep it in –20/ – 80 oC .For Retrieval Keep the vial at 35 oC for immediate thawing (in water bath) till all ice crystal dissolves .Take loop full of culture from vial & inoculate on preferred medium. 5. Lyophilization: (Freeze Drying) : Lyophilization, or freeze-drying, a form of permanent preservation, is not appropriate for all fungi In fact, the technique is used primarily with species that form numerous, relatively small propagules .This procedure is the preservation method of choice for many spore forming fungi that produce large numbers of spores 10-µm or less in diameter. Larger spores tend to collapse during the lyophilization process, and the structural damage caused is not reversible by hydration Thus, each ampoule initially must contain many viable spores

An agar slant with medium that supports good growth

and sporulation is inoculated with the organism, which is allowed to grow until it reaches the resting phase. Five or more lyophilization tubes are sterilized and labeled for immediate use. About 1.5-2.0-ml sterile menstrum(10% skimmed milk) is added to an agar slant; spores are suspended in the menstrual by gently scraping the agar

Bulletin of IAMM AP Chapter 24 Vol.17(1)

surface with a Pasteur pipette.Approximately 200 µl of the spore suspension is added to each of several lyophilization tubes. Tubes are plugged loosely with cotton, and are kept at –20to–80 oC overnight. Switch on the manifold & transfer the ampoules when the temperature remain stable between - 40 & -50 oC ,A vacuum is applied to the system till powder is formed (primary drying).Primary constriction is done to form canal of 1 mm diameter .Ampoules are fixed in manifold for secondary drying .After 2-3 hr, check vacuum by vacuum tester. The ampoules are then sealed under vacuum using a gas-oxygen torch. Vacuum must be checked again to ensure leakage or breakage.Finished lyophilization ampoules are stored in numbered plastic boxes or sealed plastic bags in a 4°C refrigerator. The purity and viability of the preparation in one lyophilization vial should be checked 1-2 weeks after preservation. Viability can be roughly categorized as poor (10-50 colonies/ampoule), moderate (50-100 colonies/ampoule),or good (100-1000colonies/ampoule)

6. Liquid Nitrogen : Storage in liquid nitrogen is an effective way to preserve many, if not most, organisms, including those that cannot be lyophilized. It costs somewhat more than lyophilization. The major advantages of liquid nitrogen storage include prevention of increased genetic variability of distributed culture stocks, timesaving, reduced labor and prevention of culture loss from contamination; and increased assurance of long-term availability of cultures. For fungal cultures that do not sporulate or that produce mycelia that grow deep into the agar, sterilized 2-ml screw-cap polypropylene vials are filled with 0.5-1.0 ml sterile 10% glycerol. Plugs 4mm in diameter are cut from vigorously growing cultures using a sterilized plastic straw. Several plugs are placed in the vial, the cap is tightened, and the tube is placed directly into the vapor phase (temperature is about -170°C) of a liquid-nitrogen tank. The accession number should be written on each cryovial. Conclusions : There is no “perfect” method of preservation appropriate for all laboratories and applicable to all fungal organisms. However, if cost and difficulty of storage are major considerations, the sterile water storage method is an excellent choice for simple storage of many commonly encountered fungi. The maximum duration of storage varies with each method and with the species being preserved, but it

generally is 10 years or less. Whenever possible, fungal strains should be preserved with one of the permanent methods (lyophilization, cryopreservation). Permanent preservation is essential for strains with critically important characteristics and for type specimens. Cultures that are permanently preserved in metabolically inactive states now can serve as type specimens, according to Article 8.4 of the International Code of Botanical Nomenclature (Greuter et al. 2000).

Collection Centers The collection centers are responsible for repository reference strains, hence creating world collections and for the maintenance of these microorganisms.

IMI (International Mycological Institute) New England;

ATCC (American Type Culture Collection) Maryland, USA;

CBS (Centralbureau Voor Schimmelcultures) Delft, Holland;

Institute For Fermentation (IFO), Osaka, Japan;

Japan Collection of Microorganismos (JMC) Wako, Saitama, Japan;

Mycotheque de L’Université Catholique de Luvain (MUCL) Luvain-le-Nueve, Belgium, and others.

References 1. Simione F, Brown EM. ATCC Preservation Methods: Freezing and Freeze-drying. 2nd ed. Rockville, Md.: ATCC; 1991. 2. Smith D, Onions AHS. The Preservation and Maintenance of Living Fungi. 2nd ed. Wallingford, Oxon, England: CABInternational; 1994.3. 3. V.K. Gupta et al. (eds.), Laboratory Protocols in Fungal Biology: Current Methods in Fungal Biology, Fungal Biology, DOI 10.1007/978-1-4614-2356-0_2, c Springer Science+Business Media, LLC 2013 4. Borman AM, Szekely A, Campbell CK, Johnson EM (2006) Evaluation of the viability of pathogenic filamentous fungi after prolonged storage in sterile water and review of recent published studies on storage methods. 5. Mycopathologia 161:361–368Smith D, Ward SM (1987) Notes on the preservation of fungi. CAB International Mycological Institute,Slough, UK

***

Bulletin of IAMM AP Chapter 25 Vol.17(1)

MDR TB : A Synopsis

Dr. J.Vijaya Lakshmi, Assistant Professor of Microbiology,

Kurnool Medical College, Kurnool

Multidrug resistant tuberculosis has become a

significant public health problem and an obstacle to

effective TB control.

Definitions: MDR TB – A TB patient whose sputum

is culture positive for MTB and is resistant in vitro to

Isoniazid(H) & Rifampicin(R) with or without other

anti TB drugs based on DST results from an RNTCP

certified C & DST laboratory. The person who shows

monoresistance to R should also be managed as if

he was an MDR TB case.

XDR TB (Extensively drug-resistant tuberculosis)

Extensively drug-resistant TB (XDR TB) is a rare type

of multidrug-resistant tuberculosis (MDR TB) that is

resistant to isoniazid and rifampin, plus any

fluoroquinolone and at least one of three injectable

second-line drugs (i.e., amikacin, kanamycin, or

capreomycin).

Causes of emergence of drug resistance:

MDR TB is a man made phenomenon. Causes can

be divided into the following:-

Microbial: Genetic mutation with an approximate

rate of 1 in 108 cell divisions resulting in drug

resistant mutants

Clinical: Inadequate treatment regimen, Improper

combination and dosages of drugs, Improper

treatment by RMPs

Patients: Poor adherence, noncompliance, adverse

drug reactions, malabsorption, substance abuse

disorder, social & economic barriers

Programmatic: Inappropriate guidelines,

inadequate training of doctors & health staff, no

monitoring of treatment , inadequate supply of

drugs.

Resistance acquired due to any reason allows the

drug resistant mutants to become the dominant

strain in a patient infected with TB.

Prevalence of MDR TB in India is about 1-2%

in new cases & 12-17% in old cases.

Laboratory aspects:

Sample collection – 2 sputum samples: 1 early

morning and 1 supervised spot are collected. But if

patient is coming from long distance or if there is a

likelihood that patient may default to give a 2nd

sample, 2 spot specimens may be collected with a

gap of 1 hour.

Transport – Samples should be transported to the

C&DST lab within 72 hrs. in cold chain using gel

packs (prefreezed at -20oC for 48hrs.) If delay is

unavoidable samples should be refrigerated.

Processing - Majority of samples are contaminated

with normal flora which rapidly overgrow the entire

surface of the medium & digest it before the

tubercle bacilli start to grow. Homogenisation and

Decontamination liquefies the organic debris &

eliminates the unwanted flora.

Smears are made for ZN staining. Two slopes of

Lowenstein Jensen medium are inoculated per

specimen with a 5mm loopful of centrifuged

sediment and incubated at 35-37oC until growth is

observed or discarded as negative after 8 weeks. In

liquid culture media (Middle Brooks medium),

growth appears as floccules with granular

nonhomogenous suspension and surface pellicle

Bulletin of IAMM AP Chapter 26 Vol.17(1)

formation.MTB grow in masses or clumps because

of the hydrophobic character of the cell surface. In

Dubos medium containing Tween 80 growth is

diffuse. Long serpentine cords are formed by

virulent strains. The growths are identified as MTB

strains by 1) susceptibility to PNB 2) positive Niacin

test. Drug sensitivity testing is done by Proportion

method.

Criteria for resistance: Any strain with 1% (critical

proportion) of bacilli resistant to any of the 4

drugs(R,H,E,S) is classified as resistant to that drug.

Methods for DST: Conventional and

nonconventional

Nonconventional methods –

1)MODS(Microscope observation direct

susceptibility), 2)Thin layer Agar method, 3) Phage

based methods(contamination rate-6%), 4)

Colorimetric redox indicator methods, 5) Nitrate

reductase assay.

Conventional methods –

a) Phenotypic DST 1) Solid – Egg based LJ media,2)

liquid – BACTEC MGIT, BACTEC9000MB,

BacT/ALERT

Reliable for H,R,S,& somewhat for E. Z is not

tested because of production of

pyrazinamidases & unstability of the drug when

incorporated into medium.

TAT (Turn around time) for solid media: 84 days

and for liquid media:7- 14 days

MGIT (Mycobacterium growth indicator tube):

Advantages- a) system alerts when tubes become

positive, b)No radioactive material used

Disadvantages- a)continous electricity required,

b)reagents are costly, c)contamination rates high,

d)further identification required if detected as

positive

b) Genotypic DST

Highly reliable for R but has limited sensitivity

for detection of H resistance.

Types:

1) DNA sequencing

2) Electrophoretic detection methods – Single

Strand Conformation Polymorphism

3) Hybridization assays – Molecular beacon,

Microassays, Membrane hybridization, Line

Probe assays.

4)PCR-RFLP for MTB based on presence of

IS6110 repetition units

5) MIRU-VNTR

6) Spoligotyping (Spacer oligotyping)

7) MTB Amplified Direct test(Gen Probe)

8) ARMS PCR(Amplification refractory mutation

system)

9) CB-NAAT(cartridge based nucleic acid

amplification testing) – Gene X pert

10) Luciferase Reporter phage assay

11) TB LAMP( Loop Mediated Isothermal

Amplification) – targets 16SrRNA

Line Probe Assays (LPA): Tests show high sensitivity

& specificity. These are a family of novel DNA strip

based tests that use nucleic acid amplification

technique & reverse hybridization methods for

rapid detection of mutation. It enables detection of

presence of MTB complex and presence of wild type

& mutation probes for resistance. If a mutation is

present in one of the target regions:-

a) The amplicon will not hybridize with the relevant

probe.

b) Mutations are therefore detected by lack of

binding to wid type probes as well as by binding to

specific probes for the most commonly occurring

mutation.

For MDR TB: Target genes – rpo B (R) , Kat G & inh

A(H)

For XDR TB: Target genes – gyr A(FQ), rrs

(Aminoglycosides), emb B (Ethambutol).

Advantages of LPA: Rapid diagnosis with TAT of 72

hrs. and increase through put.

Bulletin of IAMM AP Chapter 27 Vol.17(1)

Gene X pert:

Advantages: - Fully automated machine with 1 step

external sample preparation ; No Biosafety Cabinet

is required; There is no risk of cross contamination

as it is a closed system; Internally quality assured;

- TAT (Turn around time) is 1&1/2 hrs.

Performance: Specific for MTB, sensitivity similar to

culture.

Target gene - rpo B

TB LAMP: Contains 6 primers that target 16SrRNA.

Luciferase Reporter Phage assay:

Principle: Detection of light emmited by organism

due to activation of Luciferase gene incorporated

into the mycobacteriophage.

Prevention of emergence of MDR TB

1) Prompt diagnosis and treatment of MDR TB

2) Prevention of spread by means of Air borne

infection control measures which include-

Adequate ventilation (natural/assisted) to

ensure >12 Air Changes per hour at all times

where MDR TB & XDR TB cases stay.

Adequate sputum disposal using 5% phenol.

Cough atiquettes

Personal respiratory protection using

reusable N95 particulate respirators.

3) Implementation of a good quality DOTS

programme (FIRST PRIORITY)

4) RNTCP has started PMDT

programme(Programmatic management of

drug resistant TB) to address MDR TB problem

in India.

MDR TB with HIV coinfection : As the HIV disease

progresses & the individual becomes more

immocompromised, the clinical presentation is

proportionately more likely to be extrapulmonary

or smear negativethan in HIV uninfected TB

patients. ART should be initiated as soon as possible

in all HIV/TB coinfected patients with active TB

within 8 weeks after the start of TB treatment. For

patients who are already on ART at the time of MDR

TB diagnosis , should be continued on ART when

MDRTB therapy is initiated. Sometimes

simultaneous administration of ART & ATT may

result in Immune Reconstitution Inflamatory

syndrome(IRIS). In this there is temporary

exacerbation of signs & symptoms or radiographic

manifestations of TB after starting ATT.

Treatment regimen for MDR TB:

Intensive phase- Kanamycin, Levofloxacin, Ethambutol,

Pyrazinamide, Ethionamide, Cycloserine(6 drugs) for 6 –

9 months depending on follow up culture results

Continuation phase- Levofloxacin, Ethambutol,

Ethionamide, Cycloserine(4 drugs) for 18 months .

Treatment regimen for XDR TB:

Intensive phase- Capreomycin, Paraaminosalysilic acid,

Moxifloxacin, High dose Isoniazid, Clofazimine, Linezolid,

Amoxyclavulanic acid (7 drugs) for 6-12 months

depending on follow up culture results.

Continuation phase- PAS, Moxifloxacin, High dose

Isoniazid, Clofazimine, Linezolid, Amoxyclavulanic acid (6

drugs) for 18 months .

Chemoprophylaxis is of no role in prevention of

MDR TB & XDR TB.

Immunotherapy:

Mycobacterium Vaccae vaccination: It is postulated

that M.vaccae redirects the host’s cellular response

from a Th-2 dominant pathway to aTh-1 dominant

pathway.Th-1cells promote terminal differentiation

of macrophages into Giant cells.Th-1 cells kill the

intracellular bacilli.

Immunomodulators: TNFα for healing of cavitary

TB, TGFβ for prevention of fibrosis, Aerosolized IFN-

γ for resolution in MDR TB, Recombinant IL-2

activates the immune system & enhances the

effects of drugs in MDR TB.

Bulletin of IAMM AP Chapter 28 Vol.17(1)

NATIONAL STRATEGIES AND ALGORITHMS FOR HIV TESTING

Dr. S.Pavani,

Assistant Professor of Microbiology,

Osmania Medical College, Hyderabad

Introduction

Two distinct human immunodeficiency viruses,

HIV-1 and HIV-2 are the aetiologic agents of

AIDS. Phylogenetically, HIV-1 is divided into

Group M [10 subtypes A-K excluding I] and

Group O [9 subtypes) Group N (new virus) and

HIV-2 into 6 subtypes (A to F).

In Thailand, India and sub-Saharan Africa, ~ 90%

of HIV-1 infections are acquired through

heterosexual transmission in contrast to 10% in

the U.S. and Western Europe. Subtypes A, C and

D predominate in Africa, subtypes E and B are

commonly found in Thailand and C is the main

subtype in India; whereas, subtype B

predominates in the U.S. and Western Europe.

Laboratory diagnosis is the only method for

determining HIV status of an infected individual.

A number of tests and diagnostic kits are

available to assess the HIV status of individuals.

Serological tests are most commonly

performed.

The choice of test protocol depends upon:-

• Objectives of HIV testing

• Sensitivity of the test

• specificity of the test

• The prevalence of HIV infection among the

population

• Resources available

• Appropriateness to the strategy of testing at a

given instance

• Infrastructural facilities available

Objectives of HIV testing :

• Blood and blood products safety . This is

determined by mandatory screening of each

unit of donated blood

• Donors of sperms, organs and tissues are

tested for HIV to prevent HIV transmission to

the recipient

• Diagnosis of HIV infection in clinically

suspected individuals

• Prevention of parent to child transmission

after counseling and informed consent

• Voluntary testing: high risk groups after

counseling and informed consent

• Sentinel surveillance to monitor

epidemiological trends

• Research, surveys to identify population

groups requiring specific interventions, etc

LABORATORY DIAGNOSIS OF HIV :

A Summary of the available tests

HIV infection can be detected in the laboratory

either by detection of antibodies to HIV , or by

detection of the virus, its antigen and its DNA.

Detection of specific antigens, viral nucleic acid,

isolation / culture of virus are all confirmatory tests in

that the presence of the virus is detected. But they

are risky because of the danger of infection to

laboratory workers, are very laborious and difficult

to perform, require skilled expertise and hence are

to be done only in specified laboratories .

The indirect predictors of HIV infection (CD4 cell

count, ß2 microglobulin, etc.) are monitors of immunity

status of patients and are to be done at routine

intervals to monitor the progression of disease.

The specimens which can be utilized to detect

Bulletin of IAMM AP Chapter 29 Vol.17(1)

various markers of HIV infection are given below.

(i) Antibody detection : - Blood / serum / plasma

3-5 ml. of blood is collected in clean, screw

capped plain vial for ELISA and for the

supplemental tests. Saliva and urine have been

used to detect antibodies to HIV but the assays

have not been validated in India.

(ii) Antigen detection : - Serum / plasma; -

Cerebrospinal fluid; - Cell culture supernatant (i.e.

the tissue culture fluid)

(iii) Virus isolation : Virus Isolation can be attempted

on HIV infected tissues. It may be isolated from

blood (PBMN cells), semen, vaginal /cervical

specimen, tissue, CSF and plasma. It is less

successful on other body fluids like saliva, urine, breast

milk, tears and amniotic fluid. Virus isolation is

done for research purposes only and never for

diagnosis in India.

Detection of specific antibodies : This is done by

performing initial screening tests, which if positive, are

followed up by supplemental tests to confirm the

diagnosis.

Screening tests : The screening assays can be

either ELISA & or rapid HIV tests. ELISA is the

preferred test at blood banks, NRLs and SRLs

where a large number of samples are tested at a

time. Rapid tests are preferred at ICTCs and

PPTCTCs where the number of samples to be

testd is less and where same day/emergency

testing is required. ELISA (Enzyme Linked

Immunosorbent Assay) is the most commonly

performed test at blood bank and tertiary labs to

detect HIV antibodies.

Supplemental tests : Second and third

Elisa/Rapid; – Western blot WB is done in legal

cases, problem cases and for research.

WB is expensive, time consuming and requires

expertise to perform. This is to be done to confirm

the diagnosis on samples which given discordant

results in E/R.

Strategies/Algorithms of HIV testing :

Because of the enormous risk involved in

transmission of HIV through blood, safety of

blood products is of paramount importance.

Since the PPV is low in populations with low HIV

prevalence, WHO/GOI have evolved strategies to

detect HIV infection in different population

groups and to fulfil different objectives.

The various strategies, so designated, involve

the use of categories of tests in various

permutations and combinations.

1. ELISA/ Rapid tests: Used in strategy I, II & III

2. Supplemental test: E/R and Western

Blot(WB) : WB is used in problem cases where

discordant serological results are obtained, if

WB is not available than client should be

retested all over again after 2-4 weeks.

Strategy/Algorithm I :

Blood/Plasma/Serum is subjected once to a

highly sensitive E/R for HIV . If negative, the

sample is to be considered free of HIV and if

positive, the sample is taken as HIV infected for all

practical purposes.

This strategy is used for ensuring donation safety

(blood, organ, tissuesand sperms, etc.). Unit of

blood testing positive is destroyed as per

guidelines. A donor who gives consent to know

the result of HIV test is informed about the

result. In case the test is positive donor is

referred to ICTC for counselling, testing and

confirmation of the test result.

Strategy/Algorithm IIA :

Sample is tested as above and in case of

positive is tested further . A serum sample is

considered negative for HIV if the first ELISA or

rapid test reports it so, but if reactive, it is

subjected to a second ELISA or rapid test which

Bulletin of IAMM AP Chapter 30 Vol.17(1)

utilizes a system differen t from the first one i.e.

the principle of test and/or the antigen used is

different. It is reported reactive only if the

second ELISA/ rapid test also gives the positive

report like the first test. In case the second E/R is

non reactive, then the result is taken as

negative for sentinel surveillance purposes. This

type of HIV testing is anonymous and

unlinked.

Strategy/Algorithm IIB :

This strategy is used to determine HIV status

of clinically symptomatic suspected AIDS cases

in whom Blood/Serum/Plasma is tested with

the highly sensitive screening test. The sample is

considered negative if the test gives non-

reactive result. In case the test result is

reactive the same sample is tested with another

test kit (based on different principle of test or

having different antigens compared to the first

test), if the result is reactive with second test

kit also the sample is considered to be

positive in a symptomatic AIDS case. So, in

cases where the physician indicates that the

patient is suffering from clinical AIDS like

symptoms, the HIV status of the patient can be

confirmed as positive on the basis of two

positive test results. In case sample is positive

by first test kit and negative by second test kit,

the sample is subjected to a tiebreaker third

test. If third test is reactive, sample is reported

as indeterminate and follow-up testing is

undertaken after 2-4 weeks. In case the tie

breaker third test is negative, sample is

reported as negative. Counselling, informed

consent and confidentiality are a must in all

these cases.

Strategy/Algorithm III :

For strategy III the HIV testing done is similar as

for strategy IIB, with the added testing by a third

test for positive result.Positive confirmation of a

third reactive E/R test is required for a sample

to be reported HIV positive. If the sample gives

reactive result with two E/R and non reactive with

the third assay it is reported as “indeterminate”

and patient is called back for repeat testing after

2-4 weeks. The test utilized for the first screening

test is one with the highest sensitivity (may give

high number of false positives) and those used

for the second and third tests are those with

the highest specificity (to minimize false

positive reactions). This strategy is used for

diagnosis of HIV infection in asymptomatic

individuals at ICTCs and PPTCTCs. Counselling,

informed consent and confidentiality are a must

in these cases. Three different kits with

different antigen system and / or different

principle of test are required to follow this

strategy .

Strategy IIB & III are to be used for diagnosis

of HIV infection. Second and Third test should

be tests with the highest Positive Predictive V

alue (PPV) possible to eliminate any chances of

false positive results. If sample gives reactive

result with two assays and non reactive with

third assay in a healthy asymptomatic

individual is reported as “indeterminate” and

follow up testing is done after 2-4 weeks. A

sample reactive in first assay and non-reactive in

second assay is subjected to a third tie

breaker assay . In case the third assay also

gives non- reactive result, sample is reported

as negative for HIV infection. In such cases

however, if client history is suggestive of high

risk factors/high risk behaviour, he may be

asked to come for follow up testing after 2-4

weeks. If sample gives reactive result with

third test kit, then the individual is reported

as “intermediate” and patient is called back

for repeat testing after 2-4 weeks.

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Bulletin of IAMM AP Chapter 31 Vol.17(1)