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_Ba
cterial_i solation_characterization_and_ABST_1
https://www.researchgate.net/publication/264165875_Antimicrobial_Drug_Sensi
tivity_te
sting_and_therapeutic_use_in_Veterinary_Practice
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
cell
s
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
• Specialtransport methods for samplespacked 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
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
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 vaginaland cervical samples,
swabs are often preferred.
• Collectsamples using long
handledswabs keeping vaginal
labia wide apart.
• Careshould be taken to avoid any
contact of swab with clitoral region and
urethral opening.
• Samples/ swabs should be transportedin
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
cultur
e
• 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
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
identificati
on
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
17.
18.
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.
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
standar
d
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 regressionline analysisof
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.
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
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_characterizati
on_and
_ABST
