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Culturing of Microbiology Specimens in Laboratories

Dr.T.V.Rao MD
Professor and HOD, Dept. of Microbiology Travancore Medical College Kollam Kerala
Today there is a greater realization of importance of Microbiology laboratories in private
sector, because more patients attend the private clinics than the public run Hospitals, There are
many accredited institutes awarding post graduate and graduate qualifications in Medical
Microbiology, However few follow standard operative procedures to run the laboratories in the
private sector One should realise that an effective Microbiology laboratory has many avenues of
improving the health and future development of the hospitals .. Physicians should be confident that
the results provided by the microbiology laboratory are accurate, significant, and clinically relevant,
and that anything less is below the expected standard of care. In order to provide that level of
quality, all microbiology specimens should be properly selected, collected, and transported to
optimize analysis and interpretation
Importance of Collection of specimens -
Laboratory diagnosis of an infectious disease begins with the collection of a clinical specimen for
examination or processing in the laboratory. The person working in the laboratory should not be
confined only to the laboratory, and communicate and interact with people associated with
specimen collection, This is particularly true with the samples such as sputum, urine, vaginal and
wound swabs the Doctors working with such reports fail to realise the errors and prescribe
unnecessary Antibiotics leading to increase of Antibiotic resistance in many privately run Hospitals.
It has also been observed that the most important and frequent factor affecting laboratory analysis,
even in a well-functioning laboratory, is not the laboratory investigation itself but specimen
preparation and errors in identification or labelling. Proper collection of an appropriate clinical
specimen is, hence, the first step in obtaining an accurate laboratory diagnosis of an infectious
disease. Applying one's knowledge of microbiology and immunology for the collection,
transportation and storage of specimens is as important as its progress in the laboratory. Moreover,
the interpretation of the observation may be misleading if the specimen is inadequate or do not
contain the details of the patients clinical condition or the Antibiotics administered.

Accuracy in Collection makes difference
For specimen collection at sites that normally contain resident micro flora, care should be taken to
sample only the infected site and not the surrounding areas. For example, throat and
nasopharyngeal swabs should not touch the tongue, cheek, or saliva. Saliva is an especially
undesirable contaminant because it contains millions of bacteria, which are normal flora. Sputum,
the mucous secretion that coats the lower respiratory surfaces, especially the lungs, is discharged by
coughing or taken by a catheterization or bronchial lavage to avoid contamination with saliva. Also
the mucous lining of the vagina, cervix, or urethra can be sampled with a swabbed or applicator
stick. With good visualization of the infected areas
Testing in Laboratory
Diagnostic laboratory techniques include direct testing using a microscope, and immunological,
methods that provide immediate clues as to the identity of the microbe or microbes in the sample,
and cultivation, isolation, and identification of pathogens using a wide variety of general and specific
tests (such as from blood or other body fluids).
Gram stain
Gram staining (or Gram's Method) is a method of differentiating bacterial species into two large
groups (Gram-positive and Gram-negative).It is based on the chemical and physical properties of
their cell walls. Primarily, it detects peptidoglycan, which is present in a thick layer in Gram positive
bacteria. A Gram positive results in a purple/blue colour while a Gram negative results in a pink/red
colour. Never forget that Gram’s staining continues to be most rapid method in Diagnostic
Microbiology to improve skills in reporting and delivering timely advice to Practitioners.

Every basic Diagnostic bacteriology work involves receiving many specimens, which we categorise as
Exudates and Transudates, However nowadays we categorise the specimen as Urine, pus, wound
swabs, and catheter tips apart from ET suction materials etc. Several Agar streak plates are an
essential tool in microbiology. They allow bacteria and fungi to grow on a semi-solid surface to
produce discrete colonies. These colonies can be used to identify the organisms, isolate the strain
free of contaminants, however at times we compromise the quality by plate sharing when we streak
two or even three specimens on single plate. In order to obtain well-isolated discrete colonies, the
quadrant streak technique should be used. This allows sequential dilution of the original microbial
material (broth culture or colonies on a plate or slant) over the entire surface of a freshly warmed
plate free from moisture. As the original sample is diluted by streaking it over successive quadrants,
the number of organism’s decreases. Usually by the third or fourth quadrant only a few organisms
are transferred on the inoculating loop and these produce a few isolated colonies in the selective
media as in MacConkey agar. Isolation of pure colonies from enriched media is difficult for beginners
and accuracy is essential in identification.
The persons entrusted with culture work may follow the following instruction, to attain the
improved results in isolation and identification of bacterial pathogens
1 Flame the inoculating loop until it is red hot and then allow it to cool.
2. Remove a small amount of bacterial growth (either a loopful from a broth culture or a single
colony from a plate or slant) with the sterile inoculating loop.
3. Immediately streak the inoculating loop very gently over a quarter of the plate using a back and
forth motion.
4. Flame the loop again and allow it to cool. Going back to the edge of area 1 that you just streaked,
extend the streaks into the second quarter of the plate.
5. Flame the loop again and allow it to cool. Going back to the area that you just streaked, extend
the streaks into the third quarter of the plate
6. Flame the loop again and allow it to cool. Going back to the area that you just streaked
Extend the streaks into the centre fourth of the plate.
Incubate at defined temperature and follow the protocols of defined optimal conditions of oxygen
and carbon dioxide
Culture of Specimens
Following direct examination, clinical specimens are cultivated to generate more confirmatory data
The success of pathogen identification and treatment depends on how the specimen is collected,
handled, and stored.
Diagnostic laboratory techniques include direct testing using a microscope, and immunological or
genetic methods that provide immediate clues as to the identity of the microbe or microbes in the
Following direct testing, culturing, isolation, and identification of pathogens using a wide variety of
general and specific tests is required.
In most cases, specimens are also inoculated into differential media that define such characteristics
as fermentation patterns (mannitol salt and MacConkey agar) and as reactions in blood (blood agar).
A patient's blood is usually cultured in a special bottle of broth that can be periodically sampled for
growth. Work must be done from isolated colonies or pure cultures, as working with mixed or
contaminated cultures gives misleading and inaccurate results. From such isolates, clinical
microbiologists obtain information about a pathogen's microscopic morphology and staining
reactions, culture appearance, motility, oxygen requirements, and biochemical characteristics.
The true skills in Bacteriology remains with identifying and characterising true pathogen from the
commensal or a contaminant. If there are more than 1 type of colony are observed, and each type of
colony to be characterised according to the macroscopic and microscopic observations. A
Microbiologist can identify many rare pathogens in routine diagnostic practice with all the advancing
Automation it is very important to be dedicated to streak a plate to get the optimal results for
proper identification. Never forget Automation is a defined possibility, but we the dedicated
Microbiologists are the real hands to do wonders, and machine should
Most common difficulty experienced by many of my friends and myself is the contaminated
blood cultures, annoying to the clinician and valuable time is lost for the patients. If you are working
in a Medical college, we receive at least few specimens of blood for culturing, the diagnosis of
bloodstream infections (BSIs) is one of the most critical functions of clinical microbiology
laboratories. For the great majority of etiologic agents of BSIs, conventional blood culture methods
provide results within 48 hours; incubation for more than 5 days seldom is required when modern
automated continuous-monitoring blood culture systems and media are used. The routine media
can be used for Candia spp as they do not need other fungal special media although filamentous
fungi often require special broth media or lysis-centrifugation vials for detection. The volume of
blood that is obtained for each blood culture request (also known as a blood culture set, consisting
of all bottles procured from a single venipuncture or during one catheter draw) is the most
important variable in recovering bacteria and fungi from patients with bloodstream infections. For
adults, 20–30 mL of blood per culture set (depending on the manufacturer of the instrument) is
recommended and may require more than 2 bottles depending on the system. For children, an age-
and weight-appropriate volume of blood should be cultured. A second important determinant is the
number of blood culture sets performed during a given septic episode. Generally, in adults with a
suspicion of BSI, 2–4 blood culture sets should be obtained in the evaluation of each septic episode.
The timing of blood culture orders should be determined by patient’s clinical condition. In urgent
situations, 2 or more blood culture set scan be obtained sequentially over a short time interval, after
which empiric therapy can be initiated. In less urgent situations, obtaining blood culture sets may be
spaced over several hours or more.
The most crucial step in blood culture remains the scientific and aseptic collection of blood,
contaminated blood culture bottles are common, very costly to the healthcare system, and
frequently confusing to clinicians. To minimize the risk of contamination of the blood culture with
commensal skin flora, meticulous care should be taken in skin preparation prior to venepuncture.
Consensus guidelines and expert panels recommend peripheral venipuncture as the preferred
technique for obtaining blood for culture based on data showing that blood obtained in this fashion
is less likely to be contaminated than blood obtained from an intravascular catheter or other device.
Several studies have documented that iodine tincture, chlorine peroxide, and chlorhexidine
gluconate (CHG) are superior to povidone-iodine preparations as skin disinfectants for blood culture.
Iodine tincture and CHG require about 30 seconds to exert an antiseptic effect compared with 1.5–2
minutes. For povidone iodine preparations. CHG is not recommended for use in infants less than 2
months of age. The microbiologists should be familiar to identify the contaminants growing in the
culture plates on sub culturing in as early as less than 24 - 48 hours. However Blood cultures
contaminated with skin flora during collection are common, but contamination rates should not
exceed 3%. Laboratories should have policies and procedures for abbreviating the work-up and
reporting of common blood culture contaminants (eg, coagulase-negative staphylococci, viridians
group streptococci, diphtheroids, and Bacillus species) Clinician should contact the laboratory if the
culture result is thought to be clinically significant, do not forget all the bacteria and fungi primarily
identified need not be contaminants, and requires additional work-up and susceptibility results.
Even up to 10 % contamination rate in blood cultures are, Physicians and health care workers should
understand and support the Diagnostic Microbiology departments with scientific spirit. Or else we
cannot reduce the contamination rates.


With many years of experience in reporting the Urinary tract infections with culturing, I still feel we
have not perfected in many matters, and getting optimal conclusions are difficult and opinions vary
among the individuals when reporting, we find three or more species of bacteria in a urine
specimen, it usually indicates contamination at the time of collection and interpretation are, hence
fraught with errors. Cleaning of the Genital area carries a real priority because urine is so easily
contaminated with commensal flora, specimens for culture of bacterial urinary tract pathogens
should be collected with attention to minimizing contamination from the superficial mucosal
microbiota. Although some literature suggests that traditional skin cleansing in preparation for the
collection of midstream or “clean catch” specimens is not of benefit, many laboratories find that
such specimens obtained without skin cleansing routinely contain mixed flora and if not stored
properly and transported within one hour to the laboratory, yield high numbers of one or more
potential pathogens on culture. Interpretation of such cultures is difficult, so skin cleansing is still
recommended. We have to educate the concerned physicians, not ask the laboratory to report
“everything that grows” without first consulting with the laboratory and providing documentation
for interpretive criteria for culture that is not in the laboratory procedure manual. This is the grave
error of many private laboratories without a qualified supervision report many commensals and
contaminates as potential pathogens and testing with Antibiotic sensitivity pattern. The
differentiation of cystitis and pyelonephritis requires clinical information and physical findings as
well as laboratory information, and from the laboratory perspective the spectrum of pathogens is
similar for the two syndromes. Culturing only urines that have tested positive for pyuria, either with
a dipstick test for leukocyte esterase or other indicators of PMNs may increase the likelihood of a
positive culture, but occasionally samples yielding positive screening tests yield negative culture
results and vice versa, and undue dependency by physicians on Dip stick method should be
understood. The Gram stain is not the appropriate method to detect PMNs in urine but it can be
ordered as an option for detection of high numbers of gram-negative rods when a patient is
suspected of suffering from urosepsis. The use of urine transport media in vacuum-fill tubes or
refrigeration immediately after collection may decrease the proliferation of small numbers of
contaminating organisms and increase the numbers of interpretable results. Straight or “in-and-out”
catheterization of a properly prepared patient usually provides a less contaminated specimen
Specimens from urinary catheters in place for more than a few hours frequently contain colonizing
flora due to rapid biofilm formation on the catheter surface, which may not represent infection.
Culture from indwelling catheters is therefore strongly discouraged, but if required, the specimen
should be taken from the sampling port of a newly inserted device. We should insist that the
Clinicians should indicate whether they are sending a catheterised specimen Cultures of Foley
catheter tips are of no clinical value and will be rejected. Even we find resistance when we reject
these specimens from even the senior practitioners Collection of specimens from urinary diversions
such as ileal loops is also discouraged because of the propensity of these locations to be chronically
colonized Laboratories routinely provide antimicrobial susceptibility tests on potential pathogens in
significant numbers .Specimens obtained by more invasive means, such as cystoscopy or suprapubic
aspirations should be clearly indicated, to the laboratory, Identification of a single potential
pathogen in numbers as low as 200 cfu/mL may be significant, such as in acute urethral syndrome,
but requests for culture results reports of <10 000 cfu/mL should be coordinated with the laboratory
so that an appropriate volume of urine can reprocessed. Recovery of yeast, usually Candida spp,
even in high cfu/mL is not infrequent from patients who do not actually have yeast UTI, thus
interpretation of cultures yielding yeast is not as standardized as that for bacterial pathogens. Yeast
in urine may rarely indicate systemic infection, for which additional tests must be conducted for
Changing Trends on Detection of AFB from urine – The current peer reviewed information indicates
Recovery of Mycobacterium tuberculosis is best accomplished with first-voided morning specimens
of >20 mL, and requires a specific request to the laboratory so that appropriate processing and
media are employed. The collection of 24 hours urine is totally abandoned in future testing for AFB
detection. Never forget that genital cleaning with disinfect is essential to reduce the contamination
with the Saprophytic Acid Fast bacilli as M.smegmatis.
How to collect a specimen of urine from a young child or infant is taxing question, has few good
solutions however the method described below has scientific success
A recent paper from Madrid proposes a method to produce a flow of urine on demand in infants.
And I can report that our own unit has found it to be quite effective for both neonates, infants and
some older babies.
It takes a minimum of two people to perform this procedure. However, it is better with three, one
dedicated to making the catch.
Encourage oral fluid intake.
25 minutes following this feed, the baby/infants genitals are cleaned thoroughly with warm soapy
water and dried with sterile gauze.
Sterile container is prepared to collect specimen.
Baby is held under the armpits (just above the bed) with legs dangling (the parents can easily assist
with this).
The nurse then starts bladder stimulation which consists of gentle tapping in the suprapubic area
at a rate of 100 taps per minute for 30 seconds.
Next, the lumbar paravertebral zone (think the small of the lower back) is massaged in a light
circular motion for 30 seconds.
Step 5 and six are repeated until urine is released.
Stand clear & catch the mid-stream.
A Word of Caution – When you are running a laboratory few skilled hands under your Supervision,
1 Use commonly used media like Nutrient agar, Blood agar, Chocolate agar and Sabourouds dextrose
agar as the situation warrants
2 Use Minimal Biochemical reaction, a must is I M VI C for gram negative bacteria without fail.
3 First know what is the Normal flora in the region from where the sample is collected, or else you
will make many mistakes and confuse the clinicians and if the Physicians truly believe our results
without critical appraisal leads to misuse of Antibiotics with increasing drug resistance
4 Do not test many antibiotics to determine the drug resistance patterns, ask the Doctors what the
Antibiotic policy is and what antibiotics they prefer to use in their patents with rationalism.
Each Laboratory report will include all "legible" information, can be understood by clinician with
essential knowledge in Microbiology
Ref and Abstracts from A Guide to Utilization of the Microbiology Laboratory for Diagnosis of
Infectious Diseases:2013 Recommendations by the Infectious Diseases Society of America (IDSA) and
the American Society for Microbiology (ASM)Ellen Jo Baron,et al