The presentation summarises important methods and protocols of Clinical Microbiology. It may be useful to learners of Clinical microbiology at the undergraduate label. The presentation describes the procedures for collecting clinical samples, transport, and testing. It also describes the different methods of antimicrobial susceptibility testing and standards.

1. Clinical Microbiology (Bacteriology)
in Laboratory
BR Singh
Principal Scientist & Head of Division of Epidemiology
ICAR-Indian Veterinary Research Institute, Izatnagar-
243122, India
https://www.slideshare.net/singh_br1762/sample-collection-for-bacterial-isolation-
characterization-and-antibiotic-sensitivity-testing
https://www.researchgate.net/publication/299489486_Sample_collection_for_Bacterial_i
solation_characterization_and_ABST_1
https://www.researchgate.net/publication/264165875_Antimicrobial_Drug_Sensitivity_te
sting_and_therapeutic_use_in_Veterinary_Practice

2. Samples for Clinical microbiology
• Clinical: Excretions/ secretions/ blood/ urine/ swabs/
aspirates/ tissues/ slides
• Non-clinical
– Environmental
– Healthy companions
– Food/ feed/ water/ air

3. Errors in Sample collection for Bacterial culture
in Veterinary Clinics
• Collection of samples from poorly accessible sites
• Contamination of sample with normal
(commensal or indigenous) flora
• Failure to consider diversity of microbial agent(s)
of the disease.
• Temptation for the ease as collecting samples
• Errors in tentative diagnosis and need of
investigation.
• Lack of clinical history.
• Improper timing for sample collection.

4. What needs to be observed first when you order
a bacterial culture?
• Clinical observations suggesting infection
• Blood tests including:
– Total white blood cells (<5K and >10K) and differential leucocytes’
(Lymphocytes/ Neutrophils/ Bandemia/ left shift) counts.
– C-reactive protein (CRP) assay: Reading is >50 in serious bacterial infections.
– Procalcitonin: It is a marker of generalised sepsis.
– Markers of inflammation: Serum amyloid A, Serum ferritin, cytokines (IL-6, IL-
8, TNF-α and IL-1β), alpha-1-acid glycoprotein, Complement C3, plasma
viscosity, ceruloplasmin, hepcidin, D-dimer (a fibrin dégradation product),
fibrinogen and haptoglobin (increase in inflmmatory process and decrease in
intravascular haemolytic conditions).
– Erythrocyte sedimentation rate (ESR)
– Serology.
– Observation of any bacilli in blood smear.
• Routine urine examination: In UTI increase in pus cells.
• In Mastitis cases, slide tests for subclinical mastitis.
• For faecal samples, low grade fever, straining and stomach cramps and or
loose stool for long duration.

5. Collection of blood for culture
• At high fever stage.
• Multiple blood samples (2-3)
• From different veins.
• Local disinfection.
• In vacutainer or directly in to growth medium.
• Store and transport on ice or at 4-8oC.

6. How to sample a wound?
• Local disinfection (topical).
• Preferable sample may be fluid, cells or tissue.
• Samples should always be in duplicate.
• One for aerobic and another for anaerobic
culture.
• A sterile swab may be used to collect cells
or exudates (pus) from a superficial wound.
• From deeper wounds, aspirations of fluid into a
syringe and/or a tissue biopsy are the optimal
specimens for the recovery of pathogen.

7. Sampling for anaerobes
• Look for the clue of anaerobic infection
• Anaerobic bacteria are frequent after surgery , trauma, piercing nail
wounds.
• After prolonged antimicrobial therapy viz., aminoglycoside or broad
spectrum antimicrobial therapy and or lack of aerobic bacterial
growth.
• From wounds you may require puncturing by sterile needle and
aspirating exudates in syringe.
• To avoid oxygen to gain access to the samples or collecting sample
in vacutainer.
• Rapidly transport the sample for successful recovery of anaerobes,
and
• Special transport methods for samples packed in anaerobic-gas
packs may be desirable for detection of anaerobic bacteria.

8. Sampling from abscess
• Topical disinfection
• Drain out all the pus
• Insert a dry swab to rub on pus-forming
membrane with a few flakes of tissues and
blood.
• Swab can be transported to lab on ice or
within two hrs at ambient temperature in
sterile vials

9. Skin and mucosal samples for
bacterial culture
• A topical cleansing of the sampling area may often be
necessary .
• Dry sterile cotton-tip swab is rubbed on the suspicious
skin site, e.g., blistered or dry skin lesions or pustules, so
that something must come out of the suspected lesion.
• Moist swab are used for taking samples from mucosal
surfaces
• For collecting samples from natural orifices all care should
be taken to avoid the contact with the external openings
and for animals suitable specula can be used.
• Aspirates of fluid/pus oozing from a skin lesion using a
needle and syringe may be used.
• Skin biopsy, a small sample of skin removed under local
anaesthesia are also some times a desirable sample

10. Faecal/ Stool samples for bacterial culture
• Specify the suspected pathogen while ordering
or collecting sample for culture.
• While collecting stool samples be sure to wear
protective gloves and wash your hands
afterward.
• Never collect Faecal samples from ground or
contaminated with urine
• Better to collect directly from rectum
• Use rectal swabs for small animals.
• The faecal sample should be placed into sterile
clean, dry plastic jars with screw-cap lids may be
transported on ice and you may be required to
submit multiple samples from a patient.

11. Urine samples
• Collection of urine sample for culture is not simple in
most of the cases as you cannot instruct animals to
retract their labia or prepuce to avoid contaminants
from commensal bacteria.
• The best way to collect sample is through urinary
catheter.
• Before inserting catheter local cleansing and
disinfection is desirable.
• The sample should be collected in sterile sample
bottle and transported as soon as possible to the
laboratory.
• If long transport time is expected, transport on ice.

12. Cervical, vaginal and uterine samples
• Uterine samples can be collected either during post-
parturient infections, abortions or during oestrous
when cervix is open.
• Sheathed syringes or sheathed swabs can be used to
collect fluid or exudates present in uterus.
• Specula may be used for easier sampling.
• For deep vaginal and cervical samples, swabs are
often preferred.
• Collect samples using long handled swabs keeping
vaginal labia wide apart.
• Care should be taken to avoid any contact of swab
with clitoral region and urethral opening.
• Samples/ swabs should be transported in screw
capped containers either on ice or within two hours.

13. Collection of Samples from biopsy/
necropsy cases for culture
• Collect tissues/ exudates/ blood from heart as early as possible
after death of animal.
• Representative tissue/sample should be collected.
• For each sample separate sharp and sterile knife should be used
for cutting pieces from the most common predilection sites for
the suspected pathogen.
• Hollow organs should be swabbed with sterile cotton swabs,
preferably dry swabs.
• Hard organs like liver, kidneys etc. should be incised and sterile
swab should be inserted in to incision to soak the tissue sap.
• Samples should be sent on ice as soon as possible in sterile well
protected containers covered with non-porous wrappings.
• Proper labelling of the bottle/specimen samples is very essential.

14. Transport of samples for bacteriological
culture
• A challenge
• All the samples should be sent in the growth arresting conditions below 4oC but the
samples should not be frozen.
• Packing should be into a non-porous packaging padded with absorbent cotton or tissues.
• Swab samples can also be transported at room temperature in semisolid (gel) transport
media
• Common transport media are:
– Carry and Blair transport medium, suitable for faecal swabs;
– For fastidious and more sensitive pathogens from blood, nasal swabs, or from tissues Amies
transport medium with Charcoal;
– For fastidious organisms and anaerobes thioglycollate medium is more suitable;
– For samples suspected for yeasts Chlamydospore medium is preferred;
– For more fastidious and slow growing pathogens (Neisseria, Streptococcus equi) swabs may be
transported in Stuart medium.
– For some of the pathogens more specific medium are preferred for transport of samples on
room temperature, viz., Listeria (Listeria isolation medium), Mycobacterium (Middlebrook 7H9
broth).
– For blood culture one can directly collect sample in liquoid broth, but it is not easily available.
• If nothing is available with you, you may also send samples in sterile normal saline
having 25-30% glycerine.

15. Why do we test antimicrobial
susceptibility?
• To predict whether an infection will respond
to treatment with that antibiotic or not.
• To direct & predict antimicrobial chemotherapy.
• To review & monitor epidemiological trends.
• To set national & local antibiotic policies.
• To test the activity of a new antimicrobial agent.
• To presumptively identify isolates.

16. Components of Antibiotic Sensitivity
Testing
• 1.The identification of relevant pathogens in
exudates and body fluids collected from patients
• 2. Sensitivity tests done to determine the degree of
sensitivity or resistance of pathogens isolated from
patient to an appropriate range of antimicrobial
drugs
• 3. Assay of the concentration of an administered
drug in the blood or body fluid of patient required
to control the schedule of dosage.
16

19. Methods for antimicrobial susceptibility testing
– Direct method
• Pathological specimen
• Sensitivity result – obtained in 24 hrs.
• e.g. urine, a positive blood culture, or a swab of pus
– Indirect method
• cultured plate from pure culture
• Results after 48 hrs or more

20. How do we perform antimicrobial
susceptibility tests?
• We can use a number of methods including:-
• Disc diffusion tests - Kirby-Bauer
- Stokes’
- BSAC.
• Dilution methods: - Tube/ 96 well plate or Agar dilution
• Agar Breakpoint method
• Minimum Inhibitory Concentration (MIC) – Tube/ 96 well
plate MIC or E-tests.
• Automated methods – Vitek.
• Molecular methods – PCR.

21. Basic sets of drugs for routine susceptibility tests
Set 1 Set 2
Staphylococcus Benzyl penicillin
Oxacillin
Erythromycin
Tetracycline
Chloramphenicol
Gentamicin
Amikacin
Co-trimoxazole
Clindamycin
Intestinal Ampicillin
Chloramphenicol
Co-trimoxazole
Nalidixic acid
Tetracycline
Norfloxacin
Enterobacteriaceae
Urinary
Sulfonamide
Trimethoprim
Co-trimoxazole
Ampicillin
Nitrofurantoin
Nalidixic acid
Tetracycline
Norfloxacin
Chloramphenicol
Gentamicin
Blood and tissues Ampicillin
Chloramphenicol
Cotrimoxazole
Tetracycline
Gentamicin
Cefuroxime
Ceftriaxone
Ciprofloxacin
Piperacillin
Amikacin
Pseudomonas aeruginosa Piperacillin
Gentamicin
Tobramycin
Amikacin

25. Disc diffusion method
The Kirby-Bauer test
• Developed in the USA in 1966.
• Based on NCCLS data.
• Use Mueller-Hinton agar.
• Use standard 0.5 McFarland (BaSO4) inoculum.
• Streak inoculum in 3 directions or rotary plate.

26. Disc diffusion method : The Kirby-Bauer
test
• Use standard antibiotic-impregnated filter disc &
incubation conditions.
– 1949: Bondi and colleagues paper disks
– 1966: Kirby, Bauer, Sherris and Tuck  filter paper disks
• Demonstrated that the qualitative results of filter disk diffusion
assay correlated well with quantitative results from MIC tests
• Use standard NCCLS tables to interpret zone sizes as S, I or
R.
• Interpretation based on regression line analysis of zone
diameter size to MIC.
• Interpretation based on confluent growth of the organism.

27. Disc diffusion method : The Kirby-Bauer test
• Procedure (Modified Kirby-Bauer method: National Committee
for Clinical Laboratory Standards. NCCLS)
– Prepare approximately 108 CFU/ml bacterial inoculum in
a saline or tryptic soy broth tube (TSB) or Mueller-
Hinton broth (5 ml)
• Pick 3-5 isolated colonies from plate
• Adjust the turbidity to the same as the
McFarland No. 0.5 standard.
– Streak the swab on the surface of the Mueller-Hinton
agar (3 times in 3 quadrants)
– Leave 5-10 min to dry the surface of agar

28. Disc Diffusion Method…..
 Invert the plates and
incubate them at 35 oC,
o/n (18-24 h)
 Measure the
diameters of
inhibition zone in mm
28

29. Factors Affecting Size of Zone of Inhibition
• Inoculum density
• Timing of disc application
• Temperature of incubation
• Incubation time
 Larger zones with light inoculum and
vice versa
 If after application of disc, the plate
is kept for longer time at room
temperature, small zones may form
 Larger zones are seen with
temperatures < 35 oC
 Ideal 16-18 hours; less time does not
give reliable results

30. Factors Affecting Size of Zone of Inhibition
 Size of the plate
 Depth of the agar
medium (4 mm)
 Proper spacing of
the discs (2.5 cm)
 Smaller plates accommodate less
number of discs
 Thin media yield excessively large
inhibition zones and vice versa
 Avoids overlapping of zones

31. Factors Affecting Size of Zone of Inhibition
 Potency of antibiotic
discs
 Composition of medium
 Acidic pH of medium
 Alkaline pH of medium
 Reading of zones
 Deterioration in contents leads to
reduced size
 Affects rate of growth, diffusion of
antibiotics and activity of
antibiotics
 Tetracycline, novobiocin,
methicillin zones are larger
 Aminoglycosides, erythromycin
zones are larger
 Subjective errors in determining
the clear edge

32. Stokes’ Comparative Method
• Developed in the U.K (1972).
• A variety of media can be used including Iso-
sensitest agar (ISA), ISA & 5% lysed blood &
Chocolate ISA.
• Based on dense not confluent growth.
• Use suspension of organism in broth
equivalent in density to an overnight broth
culture.
• Inoculate fastidious organisms direct.

33. Antimicrobial Gradient Testing
E-test®
Read plates
after
recommended
Incubation
Read MIC
where elipse
intersects
scale

34. Vitek I
• The Vitek I was originally designed by NASA
for use as an on-board space exploration test
system.
• It is based on the use of small thin plastic
cards each containing many wells linked by
capillaries.
• These cards are available as susceptibility &
identification cards.

35. Molecular Methods
• Application of genotypic methods can allow rapid
detection of resistance genes direct from the sample.
• Examples include:-
• mecA gene detection by PCR denotes resistance to
methicillin in Staph aureus (MRSA).
• Rifampicin & isoniazid resistance in MDR
Mycobacterium tuberculosis can be detected using a
DNA probe.
• Antiviral drug resistance due to genetic point
mutations can be examined by PCR for HIV, CMV &
HCV.

36. Rapid tests of ABST
►Chromogenic Tests
• β lactamase enzyme- Haemophilus, Staph.
• Chloramphenicol resistance in Haemophilus
►Automated systems using new technologies
• Fluorescent methods
• Laser imaging
►Results available with MIC in 6 hrs.
►Very useful in guiding therapy.

37. • You may see the presentation at:
http://www.slideshare.net/singh_br1762/sample-collection-
for-bacterial-isolation-characterization-and-antibiotic-
sensitivity-testing
• You may read more at:
https://www.researchgate.net/publication/280934311_Sam
ple_collection_for_bacterial_isolation_characterization_and
_ABST