IDENTIFICATION OF BACTERIAL PATHOGENS

Infecti ous diseases

Infectious diseases are disorders caused by organisms — such as bacteria, viruses, fungi or parasites. Many organisms live in and on our bodies. They're normally harmless or even helpful, but some organisms under certain conditions may cause disease. Some infectious

diseases can be passed from. Types of causative agents (bacteria , virus , fungi, parasite).

Bacteria There are prokaryotes (a group of organisms whose cells lack a membrane-bound nucleus).

Many of these bacteria are saphrophytic and live by causing decay and putrification of organic material. Some eubacteria are parasitic and responsible for disease in plant, animals, and humans

Size:Size of bactreia measured by micro

The bacteria of medical importance 0.2-1.5 micron in diameter and 3-5 micron in length.Different Shapes of bacteriaCocci=spherical bacteriaBacilli=rod shaped bacteriaSpirochetes = spiral twisted bacteriaActinomycetes=branching filamentous bacteriaMycoplsma without cell memberane pleomorphic.

Structure of bacterial cell

1-cell wall2-cytoplasmic memberane

3-cytoplasm with some cytoplasmic organelles4-single circular or douple stranded DNA.

5-Some specific structurs as capsule, flagella, spores

Arrangement of bacteria :Single .

Diploid =in pairs.Streptococcus or streptobacilli = presenti n chains.Stphylococcus = present in clusters.Size:Size of bactreia measured by micro The bacteria of medical importance

0.2-1.5 micron in diameter and 3-5 micron in length.

Different Shapes of bacteriaCocci=spherical bacteriaBacilli=rod shaped bacteriaSpirochetes = spiral twisted bacteriaActinomycetes=branching

filamentous bacteriaMycoplsma without cell memberane pleomorphic.

better animal care usage appropriate

antibiotic sparing of expenses

Control of the disease preventive measures can

be initiatedAll for decrease the losses from the disease

Why We must make a specific diagnosis

♥Using the proper aseptic techniques.♥Correctly obtaining the specimen.♥Correctly handling the specimen

♥Quickly transporting the specimen to the lab.♥Once the specimen reaches the lab it is

cultured and identified

The successful identification of microbe depends on:

•Successful identification depends on how the specimen is collected, handled and stored.

•It is important that general aseptic procedures be used including sterile sample containers and sampling methods to prevent contamination of the specimen.

•What other precautions must be taken when collecting specimens?

•After collection the specimen must be taken promptly to the lab and stored appropriately (e.g. refrigeration).

Specimen Collection

•Microbiologists use 5 basic techniques to grow, examine and characterize microorganisms in the lab.

•They are called the 5 ‘I’s: inoculation, incubation, isolation, inspection and identification.

•Inoculation: to culture microorganisms a tiny sample (inoculum) is introduced into medium (inoculation).

•Isolation involves the separating one species from another.

•Incubation: once the media is inoculated it is incubated which means putting the culture in a controlled environment (incubation) to allow for multiplication.

•After incubation the organisms are inspected and identified phenotypically, immunologically or genetically.

Phenotypic Methods of Identification

1-Direct microscopical method

Microscopic Morphology include a combination of cell shape, size, Gram stain special structures e.g. endospores, granule and capsule can be used to give an initial identification.

Magnification –enhancement of size using ocular and

objective lenses.•Ocular: eyepiece (10X)

•Objective: 4X – 100X –allows for visualization of bacteria.

Gram negative

Gram positive

Ziehl Nelsen stain

2-Culture and isolation of bacteria

A bacteriological culture is a method of multiplying the bacterial cell by letting them reproduce in predetermined culture media under controlled laboratory conditions.

2-Culture and isolation of bacteria

Media classifications and functions

Enrichment•used to enhance growth of specific organisms

Supportive•support growth of most non-fastidious organisms

Selective•contains agents that inhibit the growth of all agents except that

being sought (dyes, bile salts, alcohols, acids, antibiotics)Differential

•contains factor(s) that allow certain organisms to exhibit different metabolic characteristics

1-temperature2-moisture

3-pH4-gaseous requirement

5-light

Growth requirements

Blood agar

Enriched Media

Liquid nutritive broth

Enriched Media

Mannitol salt agar

Selective media

Differential media

MacConkey’s agar

Colony characteristics

–colony form: pinpoint, circular, filamentous, irregular

–colony elevation: flat, raised ,convex

–colony margin: smooth, irregular

Biochemical tests

Many bacteria secrete enzymes as lipases , proteinases and other hydrolytic enzymes .

The microbe is cultured in a media with a special substrate and tested for an end product.

Common Tests To identify Bacterial isolates

1-Indole2-Methyl Red/ Voges Proskauer

3-Citrate4-H2S production5-Urea hydrolysis

6-Motility7-Lactose fermentation8-Sucrose fermentation

9-Glucose fermentation and gas production

Serology Serology is the scientific study of serum. In practice, the term usually refers to the diagnostic identification of antibodies in the serum. Such antibodies are typically formed in response to an infection.

Types of Antigen and Antibody reactions

1.Agglutination tests 2.Double diffusion precipitation tests

3.Immunoelectrophoresis 4.Complement fixation tests

5.Immunofluorescence testing 6.Immunoassays

•Genotypic methods involve examining the genetic material of the organisms and has revolutionized

bacterial identification and classification .•Genotypic methods include PCR (RT-PCR, RAPD-

PCR),use of nucleic acid probes, RFLP and plasmid fingerprinting .

•Increasingly genotypic techniques are becoming the sole means of identifying many

microorganisms because of its speed and accuracy.

Genotypic Methods

The responsibility of the microbiology laboratory includes not only microbial detection and isolation but also the determination of microbial susceptibility to antimicrobial agents.