general medical microbiology introduction. specimen collection the failure to isolate the causative...

General Medical General Medical MicrobiologyMicrobiology

IntroductionIntroduction

Specimen CollectionSpecimen Collection

The failure to isolate the causative The failure to isolate the causative agent of an infectious disease is agent of an infectious disease is frequently the result of frequently the result of faulty faulty collecting or transport techniquescollecting or transport techniques. . Therefore, when collecting a specimen Therefore, when collecting a specimen for microbiological examination, the for microbiological examination, the primary primary general considerationsgeneral considerations that that need to be addressed are:need to be addressed are:

General ConsiderationsGeneral Considerations

The specimen obtained must be The specimen obtained must be representative of the disease process. For representative of the disease process. For example, a throat swab can’t substitute example, a throat swab can’t substitute for sputum in cases of pneumonia. WHY?for sputum in cases of pneumonia. WHY?

Sufficient material must be collected to Sufficient material must be collected to ensure a complete and accurate exam.ensure a complete and accurate exam.

The specimen must be obtained in a The specimen must be obtained in a manner that avoids contamination by the manner that avoids contamination by the patient’s own normal florapatient’s own normal flora


If at all possible, the specimen should be If at all possible, the specimen should be obtained before initiating antibiotic obtained before initiating antibiotic therapy.therapy.

It is best to obtain the specimen at the It is best to obtain the specimen at the acute phase of the disease when the acute phase of the disease when the causative organism is most likely to be causative organism is most likely to be found in large numbers.found in large numbers.

The specimen should be delivered The specimen should be delivered promptly to the laboratory. WHY?promptly to the laboratory. WHY?


The laboratory should be provided with The laboratory should be provided with sufficient clinical information to guide the sufficient clinical information to guide the microbiologist in the selection of the microbiologist in the selection of the suitable media and incubation conditions. suitable media and incubation conditions. (Some organisms require special media (Some organisms require special media for growth, while other organisms require for growth, while other organisms require special incubation conditions).special incubation conditions).

The following specimens should normally The following specimens should normally be sterile (i.e., have no normal flora):be sterile (i.e., have no normal flora):


BloodBlood Cerebral spinal fluid (CSF)Cerebral spinal fluid (CSF) TissueTissue Serous fluidsSerous fluids Specimens from the lower respiratory tractSpecimens from the lower respiratory tract Urine taken directly from the bladder or Urine taken directly from the bladder or

kidneykidney The following specimens are likely to be The following specimens are likely to be

contaminated with normal flora:contaminated with normal flora: Specimens from the upper respiratory tract, Specimens from the upper respiratory tract,

including the mouth and noseincluding the mouth and nose


SputumSputum FecesFeces Genital tract specimensGenital tract specimens SkinSkin

The following specimens may be The following specimens may be contaminated with normal flora, but the contaminated with normal flora, but the numbers of contaminating organisms numbers of contaminating organisms are likely to be quite low:are likely to be quite low: Conjunctiva of the eyesConjunctiva of the eyes External earsExternal ears

Circumventing Normal FloraCircumventing Normal Flora

There are several different ways that There are several different ways that one may use to circumvent problems one may use to circumvent problems of contamination with indigenous of contamination with indigenous normal flora:normal flora: Antisepsis – antiseptics such as tincture Antisepsis – antiseptics such as tincture

of iodine should be applied to the skin of iodine should be applied to the skin prior to aspiration of abscesses and prior to aspiration of abscesses and various normally sterile body fluids such various normally sterile body fluids such as blood and CSFas blood and CSF


Decontamination – procedures that Decontamination – procedures that selectively inhibit or destroy selectively inhibit or destroy microorganisms other than those of microorganisms other than those of specific interest may be used. For specific interest may be used. For example sputum may be treated with example sputum may be treated with NaOH prior to culturing for mycobacteriaNaOH prior to culturing for mycobacteria

Use of selective media – use media that Use of selective media – use media that selectively inhibits growth of normal selectively inhibits growth of normal flora and allows for growth of flora and allows for growth of pathogens, i.e. MSA (more on this later)pathogens, i.e. MSA (more on this later)


Quantification – the classic example of Quantification – the classic example of quantification procedures is the quantification procedures is the procedure used for urine specimens. A procedure used for urine specimens. A calibrated loop that delivers 1 ul of urine calibrated loop that delivers 1 ul of urine (1/1000 of a ml) is used and if the (1/1000 of a ml) is used and if the colony count is > 100 colonies, the colony count is > 100 colonies, the result is considered to be significant and result is considered to be significant and indicative of infection (How many indicative of infection (How many organisms would this be/ml?)organisms would this be/ml?)

Urine colony countsUrine colony counts


Microscopy –Microscopy – A cytological exam should be done to look A cytological exam should be done to look

for the presence of squamous epithelial cells for the presence of squamous epithelial cells in urine, sputum, or wound specimens in urine, sputum, or wound specimens which, when present, indicate likely which, when present, indicate likely contamination with skin or mucosal flora. contamination with skin or mucosal flora.

A new specimen should be requested when A new specimen should be requested when numerous squamous epithelial cells are numerous squamous epithelial cells are present in the specimen.present in the specimen.

Inappropriate sputum specimenInappropriate sputum specimen

Good sputum specimenGood sputum specimen


Invasive procedures that allow the Invasive procedures that allow the physician to avoid normal flora in physician to avoid normal flora in collecting the specimen including:collecting the specimen including: Transtrachael aspirateTranstrachael aspirate Suprapubic aspirateSuprapubic aspirate BronchoscopyBronchoscopy Needle biopsiesNeedle biopsies

Transtrachael aspirateTranstrachael aspirate

Suprapubic aspirateSuprapubic aspirate

General instructions for General instructions for collection of specimenscollection of specimens General instructions for specimen collection:General instructions for specimen collection:

Identification – the specimen should be labeled with Identification – the specimen should be labeled with the following:the following: Patient’s name and identification numberPatient’s name and identification number Patient’s locationPatient’s location Patient’s physicianPatient’s physician Site/source of specimenSite/source of specimen Type of exam requested (bacterial, fungal, viral, parasites)Type of exam requested (bacterial, fungal, viral, parasites) Tentative diagnosis (Why is this important?)Tentative diagnosis (Why is this important?) Date and time of specimen collection (Why is this Date and time of specimen collection (Why is this

important?)important?) If antibiotics have been administered the type, dosage and If antibiotics have been administered the type, dosage and

time given should be providedtime given should be provided

General instructions for General instructions for collection of specimenscollection of specimens

Transport containers, devices, media:Transport containers, devices, media: Swabs are convenient and economical, but:Swabs are convenient and economical, but:

They are often inadequate in terms of the amount They are often inadequate in terms of the amount of specimen required for multiple types of of specimen required for multiple types of culturing.culturing.

The recovery of bacteria is usually< 10% of the The recovery of bacteria is usually< 10% of the original inoculum. original inoculum.

Swabs are often used for throat cultures and for Swabs are often used for throat cultures and for cervical, vaginal and urethral secretions. cervical, vaginal and urethral secretions.

They should not be used when pus or exudate is They should not be used when pus or exudate is available, for surgical specimens, or for cultures available, for surgical specimens, or for cultures for anaerobes or for anaerobes or mycobacteria.mycobacteria.


Syringes – these are good for aspirates. The tip of the Syringes – these are good for aspirates. The tip of the needles should be plugged with a sterile stopper or the needles should be plugged with a sterile stopper or the needle should be removed and the syringe capped.needle should be removed and the syringe capped.

Tubes, bottles, and jars should be sterile and leak proof.Tubes, bottles, and jars should be sterile and leak proof. Fecal transport systems – polyvinyl alcohol fixative Fecal transport systems – polyvinyl alcohol fixative

should be used for preservation of fecal parasitesshould be used for preservation of fecal parasites For sexually transmitted diseases it is best to inoculate For sexually transmitted diseases it is best to inoculate

media directly at the bedside of the patient or to use a media directly at the bedside of the patient or to use a swab/transport media system specifically designed for swab/transport media system specifically designed for optimal recovery of sexually transmitted pathogens.optimal recovery of sexually transmitted pathogens.

For specimens for viral examination or for anaerobic For specimens for viral examination or for anaerobic culturing, appropriate transport media should be used.culturing, appropriate transport media should be used.


The specimen must be promptly transported to The specimen must be promptly transported to the laboratory to preserve the viability of the laboratory to preserve the viability of fastidious organisms and to prevent fastidious organisms and to prevent overgrowth of fastidious organisms by more overgrowth of fastidious organisms by more rapidly growing bacteria which may be rapidly growing bacteria which may be insignificant. insignificant. Sometimes refrigeration of the specimen is Sometimes refrigeration of the specimen is

warranted (i.e. urine specimens), warranted (i.e. urine specimens), Sometimes refrigeration will kill fastidious organisms Sometimes refrigeration will kill fastidious organisms

(i. e. (i. e. Streptococcus pneumoniaeStreptococcus pneumoniae or or Neisseria Neisseria gonorrhoeae gonorrhoeae from sputum or genital tract specimens, from sputum or genital tract specimens, respectively)respectively)

Specific guidelinesSpecific guidelines

Specific guidelines for specimen collection:Specific guidelines for specimen collection: SepticemiaSepticemia means that organisms, or their means that organisms, or their

toxins are present, and growing in the blood. toxins are present, and growing in the blood. BacteremiaBacteremia simply means the presence of simply means the presence of

organisms in the blood without causing infection organisms in the blood without causing infection (Can you think of examples where you might (Can you think of examples where you might find this?) find this?)

For septicemia, 2-3 cultures should be collected For septicemia, 2-3 cultures should be collected by venipuncture in a 24 hour periodby venipuncture in a 24 hour period 20-30 mls should be collected for each sample. 20-30 mls should be collected for each sample. The samples should be inoculated into culture media The samples should be inoculated into culture media

directly at the bedside of the patient.directly at the bedside of the patient.

VenipunctureVenipuncture


Infections of other normally sterile body fluidsInfections of other normally sterile body fluids Meningitis and encephalitis – collect CSF via a lumber Meningitis and encephalitis – collect CSF via a lumber

puncturepuncture Pleural, pericardial and synovial fluids – aspirate and Pleural, pericardial and synovial fluids – aspirate and

collect sufficient quantities for a thorough exam.collect sufficient quantities for a thorough exam.

Wounds Wounds The best specimens are aspirates of pus or exudate. The best specimens are aspirates of pus or exudate. A swab is usually not a good way to collect A swab is usually not a good way to collect

specimens from wounds.specimens from wounds.

Lumbar punctureLumbar puncture


Upper respiratory tract infections – a Upper respiratory tract infections – a swab is sufficient for collecting URT swab is sufficient for collecting URT specimensspecimens

Lower respiratory tract infections – Lower respiratory tract infections – collect sputum. Alternatively, may use:collect sputum. Alternatively, may use: Transtrachael aspiration, Transtrachael aspiration, Bronchial wash, or Bronchial wash, or Lung puncture for collecting the specimen.Lung puncture for collecting the specimen.

Urinary tract infectionUrinary tract infection Use a clean voided midstream specimenUse a clean voided midstream specimen

Clean voided midstreamClean voided midstream


Use catherizationUse catherization Use suprapubic aspiration of bladder or kidneyUse suprapubic aspiration of bladder or kidney

Gastroenteritis – collect a stool sample in a Gastroenteritis – collect a stool sample in a sterile container. sterile container. For intestinal parasites, three separate specimens For intestinal parasites, three separate specimens

should be collected because some organisms are should be collected because some organisms are only present intermittently.only present intermittently.

Genital tract infections – a swab or aspirate of Genital tract infections – a swab or aspirate of exudate plus direct inoculation on media is exudate plus direct inoculation on media is bestbest

Ocular infections – a swab is sufficientOcular infections – a swab is sufficient Tissue specimens – that should be obvious!Tissue specimens – that should be obvious!

Direct examDirect exam

Direct examination of specimensDirect examination of specimens Gross examination of the specimen may yield Gross examination of the specimen may yield

important information:important information: If CSF is cloudy this probably indicates infectionIf CSF is cloudy this probably indicates infection Sputum – the color, consistency and odor may give Sputum – the color, consistency and odor may give

clues as to the causative agent of infectionclues as to the causative agent of infection If the stool contains mucous and blood, this is typical of If the stool contains mucous and blood, this is typical of

dysenterydysentery If the odor is foul, the specimen may contain anaerobesIf the odor is foul, the specimen may contain anaerobes Visible granules (which are aggregates of Visible granules (which are aggregates of

actinomycetes) are found in actinomycete infectionsactinomycetes) are found in actinomycete infections

Initial microscopic Initial microscopic examinationexamination Initial microscopic examinationInitial microscopic examination

Differential stainsDifferential stains Gram stain – Gram stain –

Allows one to determine if organisms are Gram positive or Allows one to determine if organisms are Gram positive or Gram negative (Why would this be important for a physician?)Gram negative (Why would this be important for a physician?)

To determine the shape of bacteria it is usually necessary to To determine the shape of bacteria it is usually necessary to use oil. On low power it is possible to see fungi, some parasites use oil. On low power it is possible to see fungi, some parasites and WBCs. (The and WBCs. (The hallmarkhallmark of an acute bacterial infection is of an acute bacterial infection is numerous PMNs). numerous PMNs).

Positive and negative controls should always be done (Why?)Positive and negative controls should always be done (Why?) A Gram stain of a direct smear can provide important A Gram stain of a direct smear can provide important

information for some specimens, but is useless for others (Give information for some specimens, but is useless for others (Give examples of each)examples of each)

It is important to not over interpret Gram stain resultsIt is important to not over interpret Gram stain results

Gram stain of Gram stain of Bacillus Bacillus speciesspecies

Gram stain of Gram stain of Staphylococcus Staphylococcus aureusaureus

Gram stain of Gram stain of Neisseria speciesNeisseria species

Gram stain of Gram stain of Haemophilus Haemophilus speciesspecies

Initial microscopic Initial microscopic examinationexamination

Acid fast stain – Acid fast stain – Divides organisms into acid-fast and non acid-fast Divides organisms into acid-fast and non acid-fast

organismsorganisms Acid fast staining results are clinically very Acid fast staining results are clinically very

important for diagnosing tuberculosis. important for diagnosing tuberculosis. Mycobacteria tuberculosisMycobacteria tuberculosis grows so slowly that it grows so slowly that it may be 6-8 weeks before a culture report is may be 6-8 weeks before a culture report is signed out. It is important that the physician signed out. It is important that the physician know if acid-fast bacilli are seen in the direct know if acid-fast bacilli are seen in the direct smear so that appropriate antimicrobic therapy smear so that appropriate antimicrobic therapy can be initiated as soon as possible.can be initiated as soon as possible.

Acid fast stainAcid fast stain


Special stainsSpecial stains Spore stain The position of the spore may be Spore stain The position of the spore may be

diagnostically importantdiagnostically important


Capsule stain – usually the background and Capsule stain – usually the background and the organism are stained while the capsule the organism are stained while the capsule is left unstained.is left unstained.


Trichrome stain – for permanent stained Trichrome stain – for permanent stained smears of intestinal parasites:smears of intestinal parasites:


Iron-hematoxylin stain – another way to Iron-hematoxylin stain – another way to make make permanent stained smears of make make permanent stained smears of intestinal parasitesintestinal parasites


Wrights and Giemsa stains of blood – Wrights and Giemsa stains of blood – parasites and bacteria in the blood may be parasites and bacteria in the blood may be seenseen


Wet mountsWet mounts India ink mount for India ink mount for Cryptococcus Cryptococcus

neoformansneoformans


Lactophenol cotton blue – to observe fungiLactophenol cotton blue – to observe fungi


10% KOH – to observe fungi from skin 10% KOH – to observe fungi from skin scrapings. The KOH destroys the epithelial scrapings. The KOH destroys the epithelial cells without harming the fungal elements.cells without harming the fungal elements.


Iodine stain – used for stool examination for Iodine stain – used for stool examination for parasitesparasites

general medical microbiology introduction. specimen collection the failure to isolate the causative...

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