Workflow Bactriology

Contents
1.
General Guidelines
2.
General Workflow
17
3.
Specimen collection, transport & processing

3.1. Anaerobic
3.2. CSF
3.3. Bone and Tissue
3.4. Eye
3.5. Contact Lens
3.6. Ear
3.7. Pus and Swab
3.8. CAPD
3.9. Catheter Tip
3.10. Urine
3.11. Fecal
A.
4.
References
76
5.
Appendices
85
1. GENERAL GUIDELINES
This is a comprehensive standard operative procedure manual for all types of specimens received in a
clinical bacteriology diagnostic laboratory serving a tertiary care hospital. The manual has been
compiled by referring to international protocols customized to the needs and the infrastructure already
available in India or infrastructure that can be achieved by upgradation. Both conventional and
automated procedural alternatives are included. It is intended that all participating laboratories,
including Nodal Centers and the Regional Center laboratories, will strictly adhere to the procedures.
The manual has been organized to place each part of the procedure together, including collection,
specimen processing, supplies, QC, and step-by-step testing procedure. This will allow the user to see
an overview of the entire procedure together. Guidelines for specimen collection and transport can be
separately made available to the collection points and those for processing are to be made available in
the processing laboratories. All the laboratories must isolate, identify to species level and carry out
susceptibility tests of significant bacterial isolates as per guidelines provided. For isolates, which are
difficult to identify, Regional Center laboratories can send the isolates to Nodal Centers for further
characterization.
2.0 General Workflow - Microbiology Laboratory
2.0 General Work Procedure Microbiology Laboratory

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
This high level work procedure is applicable for processing internal and
external specimens in Microbiology Laboratory.
The Medical Laboratory Technologist (MLT) will receive bar coded
specimens from the Common Receiving Area (CRA) and will sort out to
individual Microbiology section.
The MLT or Scientific Officer (SO) /Doctor (Dr) will acknowledge the
receipt of the specimen at the individual Microbiology section.
The MLT or SO or Dr will check and verify the test request and the specimen.
If the specimen is not suitable for processing, the MLT or SO or Dr shall
reject request and the reason for rejection shall be given.
For the group and batch tests, system has provision to create Work list.
Analysis of the sample shall be proceeded either by automated system
which is interfaced or by manual method.
All preliminary reports shall be documented. The positive preliminary
report shall be verified and validated by SO or Dr before being viewed by
the care provider. The analyzer will release automatically all negative
preliminary results through the CIS.
This preliminary report supercedes any previous preliminary result.
The test shall be repeated for all unsatisfactory preliminary performance.
The MLT shall proceed with the test after satisfactory performance has been attained.
The result shall be documented. If any supplementary test is required, the
MLT/SO/Dr will add on the related test for further investigation. Refer to
LIS/HLWP/Path/3 for any test that needs to be out sourced
Upon completion, the MLT /SO/Dr shall verify all the final results. Any
unsatisfactory performance of the test shall be repeated.
All final results shall be validated by MLT/SO/Dr.
For external requests, the final results shall be released together with the List of External
Pathology Report (system generated) into the secured respective pigeon holes. Whereby
for in house request, final results can be viewed through the Clinician Information
System.
3.0 Specimen collection, transport & processing

3.1 Flow Chart for Identification of Anaerobic Bacteria
1. Label
2. Record
Received specimen for anaerobic

(Sterile container)
Grams stain
Culture
Anaerobic Blood agar

(AnO2)
Incubate 35 2 C 48 hrs.
No growth
Read media
Growth
Identification Test
Report
Mixed growth
3.1
Work Procedure for Identification of Anaerobic Bacteria
Day 1
3.1.1. Collect the samples from the specimen reception.
3.1.2. Check the sample and the request form.
3.1.2.1.
If not complete or have any discrepancy of the sample and

request form, reject the request. The criteria of specimen
rejection refer to core document.
3.1.2.2.
Incomplete request form must be stamped with
INCOMPLETE in red ink at centre top of the request form.
3.1.2.3. If the sample in acceptable criteria, record the patient data
into respective Record Book and specimen is given unique lab
reference number
3.1.3. Label the culture plates (aerobic and anaerobic blood agar) with
unique specimen number and date perform.
3.1.4. Inoculate the specimen onto culture media. If the specimen is liquid,
transfer 1 to 2 drops on culture media.
3.1.5. Place 1 drop of specimen or make smear from tissue or pus onto clean
glass slide.
3.1.6. Aseptically streak each isolation media to produce isolated colonies.
Place a metronidazole disc at the primary streak of anaerobic blood
agar.
3.1.7. Incubate the plates at 35 2 C for 48 hours in anaerobic jar. Prepare
anaerobic condition by activating the anaerobic gas pack and insert
either chemical indicator or biological indicator (culture Pseudomonas
aeruginosa onto anaerobic blood agar) to indicate anaerobic condition.
3.1.8. Aerobic culture agar is incubated aerobically at 35 2 C for 48 hours.
Day 3
3.1.9. Examination of Culture Plates
3.1.9.1.
Observed for any growth.If no growth. Report as No

growth obtained for anaerobic culture in the worksheet.
3.1.9.2.
If there is growth, observed the morphology of the
colonies. If the same colonies grew on aerobic Blood agar, it is
not an anaerobic bacteria. Make sure there is zone of inhibition
around the metronidazole disc, as most of anaerobic bacteria are
susceptible to that disc.
3.1.9.3.
If there is mixed growth on anaerobic blood agar, purify by
subculturing onto another anaerobic blood agar.
3.1.9.4.
Record morphology of each colony type, such as pitting,

swarming, hemolysis, pigment, greening of the medium, etc.
3.1.9.5.
Make a smear for Gram stain on suspected colony. You
may use basic fuchsin in place of safranin to enhance the staining
of some gram negative organisms. Examine the smear for
presumptive identification.
3.1.9.6.
You may need to subculture suspected colony on CHOC
agar and incubate at 35 2 C in a 5 % CO2 condition for 24 to
48 hours to detect slow growing aerobic organisms such as
Capnocytophaga, Actinobacillus and Eikenella species
(aerotolerance testing).
3.1.10. Identification of Anaerobic organisms.
3.1.10.1.
Perform basic test such as catalase test, oxidase test and
other test such as culture on bile esculin agar, sugar assimilation
test (Glucose, Lactose, Maltose and Sucrose), indole, urea,
motility and etc.
3.1.10.2.
Perform identification by using commercial identification
kit. Use young culture (24 to 48 hours) and adequate colonies for
optimum result.
3.1.10.3.
Incubate the kit in an appropriate condition as
recommended by the manufacturer.
3.1.10.4.
Antimicrobial Susceptibility Testing (AST) performed on
request or when there is a need.
3.1.10.5.
Record result into worksheet and respective record book.
3.1.10.6.
Result shall be validated by Scientific Officer or Clinical
Microbiologist before being dispatch.
3.2 Flow Chart for Processing of CSF Specimen
1. Label
2. Record
Received specimen for CSF

(sterile container)
Macroscopic Examination
Microscopic Examination
Cell Count
Grams stain
India Ink
Latex Agglutination
Culture
BA
MAC
CHOC
SDA
Incubate 35C (2)

24hrs
Re-incubate
Overnight
Read media
No growth
Growth
Mixed
Identification Test
Sensitivity Test
Report
3.2.
Work Procedure for Processing of CSF Specimen
Day 1
3.2.1 Collect the samples from the specimen reception.
3.2.2 Check the sample and the request form:
3.2.2.1 If not complete or have any discrepancy of the sample and
request form, please inform to the respective ward.
3.2.2.2 Incomplete request form must be stamped with
INCOMPLETE in red ink at centre top of the request form.
3.2.2.3 If the sample in acceptable criteria, record the patient data
into CSF Record Book and specimen is given a unique lab
reference number.
3.2.3
MACROSCOPIC EXAMINATION
3.2.3.1 Observe the appearance of the CSF specimen by colour and
the presence of turbidity, deposit or clot.
e.g: CSF appearance: clear/ xanthrochromic/ cloudy/
turbid/blood stained
3.2.4
INITIAL PROCESSING OF SPECIMEN

3.2.4.1 Label the culture plate with unique specimen barcode number
and date perform.
3.2.4.2 Label 2 clean glass slides with Lab No.
3.2.4.3 Transfer 2 drop of CSF into sterile test tube and add 2 drop of
Methylene Blue. Leave for 10 minutes before performing cell
count.
3.2.4.4 Aseptically transfer CSF (1.0 ml 1.5 ml) into a sterile
microtube.
3.2.4.5 Centrifuge at 3,000 rpm for 5 minutes.
3.2.4.6 Aseptically remove the supernatant into a sterile test tube.
Double boil the supernatant for 5 minutes at 100C. Set the
timer.(Bacterial antigen)
3.2.4.7 Transfer 1 drop of CSF sediment onto each BA, MAC, SDA
and CHOC agar.
3.2.4.8 Also transfer 1 drop of CSF sediment onto 2 clean slides for
Gram Stain and India ink.
3.2.4.9 Spread the drop on the slide to make a smear for Gram Stain.
3.2.4.10 Make a wet smear for India ink.
3.2.5
MICROSCOPIC EXAMINATION
3.2.5.1 Cell Count
a. Transfer CSF-Methylene Blue preparation into Kova
Glasstic Slide and perform cell count for Lymphocytes &
polymorphonuclears (Neutrophils).
b. Count number of both cells according to KOVA insert (Low

or High cell count)
c. Interpret the actual number of cells presence by referring to
the Kova Glasstic Slide 10 with Grids Value Table.
d. Report Total Cell Count (TWBC) and then calculate the
percentage (%) of Lymphocytes & polymorphonuclears.
e.g.:
TWBC = 8 cells/l
Lymphocytes = 62.5% (5/8 x 100)
Neutrophils = 37.5% (3/8 x 100)
e. Cell count not performs for bloody sample and report as

unsuitable for testing.
3.2.5.2.
Grams Stain
Read smear for Grams stain and report either No organism
seen or presence of any organism.
e.g.:
Gram positive cocci/ bacilli or
Gram negative cocci/ bacilli
3.2.5.3.
India ink
Read smear for India ink and report either negative or positive
for Cryptococcus.
3.2.6. LATEX AGGLUTINATION

3.1.6.1 When the timer went off, take out the supernatant and do latex
agglutination test using Latex Bacterial Antigen Kit. Refer to
kit insert file.
3.1.6.2 Report result based on agglutination against any of 5 antigens
listed in the test.
3.2.7. REPORTING THE INITIAL RESULT
3.1.7.1 Record the result of cell count, Grams stain, India Ink and
Latex test into record book and on the test request form (PERPAT 301).
3.1.7.2 Call the ward to inform Microscopic (cell count, Grams stain
and India ink) and latex test result.
3.1.7.3 Record the name of the wards staff that received the call, the
time, date and your name into the record book.
3.2.8. CULTURE AND INCUBATION
3.2.8.2.
Aseptically streak the drop on the culture media to produce

single colonies.
3.2.8.3. Incubate the plates at 35 2 C for 18-24 hours. Chocolate
agar is incubated in enriched CO2 condition.
Day 2
3.2.9. After incubation (18-24 hours), examine the BA, MAC, CHOC and
SDA media, if no growth, re-incubate and examine after overnight
incubation.
3.2.10. If no growth after 48 hours incubation, report Culture: No growth was
obtained.
3.2.11. If growth is present on the plates, record the colonys morphology and
quantity of each colony type e.g. 1+, 2+, or 3+ (few, moderate or
heavy).
3.2.12. Any mixed growth culture more than 2 types of colonies perform
minimal ID. No sensitivity test required. Record the results into
request form.
3.2.13. If growth is pure colonies or not more than 2 types of colonies,
perform identification and sensitivity according to Gram positive or
Gram negative identification procedure using Vitek 2 Compact.
3.2.14. Incubate the respective biochemical identification media/kit and
sensitivity media in appropriate condition.
Day 3
3.2.15. Read the Biochemical identification media/kit and sensitivity plate.
3.2.16. Record all the results into microbiology worksheet, sensitivity form,
and CSF Record Book completely.
3.2.17. Attach the sensitivity form to the request form (PER-PAT 301)
3.2.18. Place all completed reports into respective drawer.
3.2.19. All reports will be validate by microbiologist before being dispatch
3.2.20. Any discrepancy, refer to the microbiologist.
3.2.21. The copy of request form shall be attached with microbiology
worksheet and must be kept in designated place.
3.3 Flow Chart for Processing of Bone & Tissue Specimen
1.
Received specimen in sterile

container
Label
Recor
2.
Add sterile tryptone water into

the specimen
Grams stain
10.0
AFB stain (if indicated)
Culture
Incubate at 35 2 C
Reincubate plates
after 24 hours
incubation.
Read Plate
No growth
Growth
Growth
Identification Test
No growth
Sensitivity Test
Report
BA
MAC
Francis Media
BA (Ano2)
CHOC
SDA
3.3 Work Procedure for Processing of Bone & Tissue Specimen

Day 1
3.3.1 Collect the samples from the specimen reception.
3.3.2 Check the sample and the request form.
If not complete or have any discrepancy of the sample and request form,
reject the request. The criteria of specimen rejection refer to core
document.
Incomplete request form must be stamped with INCOMPLETE in red
ink at centre top of the request form.
If the sample in acceptable criteria, record the patient data into
Pus/Bone/Tissue/Swab/Tip Record Book and specimen is given a unique
lab reference number.
3.3.3
Label the culture plate with unique specimen number and date perform.
3.3.4
Aseptically mixed some sterile tryptone water with the specimen and leave it
for 5 minutes.
3.3.5
The mixture of bone or tissue is then inoculated onto respective culture media.
Aseptically streak each isolation media to produce single colonies. Incubate
the plates at 35 2 C for 18-24 hours. Chocolate agar is incubated in enriched
CO2 condition.
3.3.6
All specimens are cultured into anaerobic blood agar.
3.3.7
A smear is then made onto a clean, labeled slide for Grams stain.
3.3.8
Ziehl-Neelson stain is done if requested/clinically indicated suspicion of

Mycobacterium infection.
3.3.9
If Gram stain showed Gram positive bacilli and Ziehl Neelson stain showed
AFB is present, it is very likely that Mycobacterium species is present.
3.3.10 Give specimen to TB Laboratory to culture onto Lowen-Jensen media.

Specimen will process to recover any Mycobacterium present and inform Pus
section staff of the result.
3.3.11 Record the Gram stain result onto Pus/Bone/Tissue/Swab/Tip Record Book.
3.3.12 Keep Gram stain slides into the designated slide box.
Day 2
3.3.13 After incubation (18-24 hours), examine the BA, MAC and other plates, if no
growth, re-incubate and examine after overnight incubation.
3.3.14 If no growth after 48 hours incubation, report Culture: No growth was
obtained.
3.3.15 If growth is present on the plates, record the colonys morphology and
quantity of each colony type e.g. 1+, 2+, or 3+ (few, moderate or heavy).
3.3.16 Any mixed growth culture more than 2 types of colonies identify according to
colonys morphology. No sensitivity test required. Record the results into
request form (PER-PAT 301), microbiology worksheet and follow the
instruction in point 9.3 number 2 to number 7.
3.3.17 If growth is pure colonies or not more than 2 types of colonies, perform
identification and sensitivity according to Gram positive or Gram negative
identification procedure.
3.3.18 Incubate the respective biochemical identification media/kit and sensitivity
media in appropriate condition.
Day 3
3.3.19 Read the Biochemical identification media/kit and sensitivity plate.
3.3.20 Record all the results into microbiology worksheet, sensitivity form, and
Pus/Bone/Tissue/Swab/Tip Record Book completely.
3.3.21 Attach the sensitivity form to the request form (PER-PAT 301).
3.3.22 Place all completed reports into respective drawer.
3.3.23 All reports will be validate by microbiologist before being dispatch
3.3.24 Any discrepancy, refer to the microbiologist.
3.3.25 The copy of request form shall be attached with microbiology worksheet and
must be kept in designated place.
3.3.
Flow Chart for Processing of Eye Specimen
Received specimen for Eye Swab

(in transport medium)
Grams stain
BA
MAC
CHOC
SDA
Culture
Incubate 35 2 C
Read media
No growth
Mixed growth
Growth
Identification Test
Sensitivity Test
Report
1. Label
2. Record
3.4. Work Procedure for Processing of Eye Specimen

Day 1
3.4.2.1.
If not complete or have any discrepancy of the sample and request

form, reject the request. The criteria of specimen rejection refer to
core document.
3.4.2.2. Incomplete request form must be stamped with INCOMPLETE
in red ink at centre top of the request form.
3.4.2.3. If the sample in acceptable criteria, record the patient data into Eye
& Ear Record Book and specimen unique number is given.
3.4.2.4. Label the culture plate with unique specimen number and a date
perform.
3.4.2.5. Inoculate the specimen onto respective culture media.
3.4.2.6. Aseptically streak the inoculum to produce single colonies.
3.4.2.7. Incubate all the culture plate at 35 2 C for 18-24 hours.
3.4.2.8. A smear is then made onto a clean slide for Grams stain.
3.4.2.9. Record the Gram stain result onto Eye & Ear Record Book.
3.4.2.10. Keep the Gram stain slides into the designated slide box for
Microbiologist to verify.
Day 2
3.4.3. After incubation (18-24 hours), examine the BA, MAC, CHOC and SDA, if
no growth, re-incubate and examine after overnight incubation.
obtained.
3.4.6. Any mixed growth culture more than 2 types of colonies identify according to
request form and microbiology worksheet
3.4.7. If growth is pure colonies or not more than 2 types of colonies, perform
identification procedure (reference).
3.4.8. Work instructions for sensitivity test.
3.4.9. If yeast is grown, give plate to Mycology unit. Mycology staff will record
result in Mycology record book and Eye & Ear record book.
3.4.10. Incubate the respective biochemical identification media/kit and sensitivity
Day 3
3.4.12. Record all the results into microbiology worksheet, sensitivity form, and Eye
& Ear Record Book completely.
3.4.13. Attach the sensitivity form to the request form.
3.4.15. All reports will be validated by microbiologist before being dispatch.
3.4.17. The copy of request form shall be attached with microbiology worksheet and
3.5 Flow Chart for Processing of Contact Lens Specimen
Received specimen for Contact

lens (contact lens container)
BA
MAC
CHOC
SDA
Culture
Grams stain
Incubate 35 2 C
Read media
Mixed growth
No growth
Growth
Identification Test
Sensitivity Test
Report
1. Label
2. Record
3.5 Work Procedure for Processing of Contact Lens Specimen

Day 1
3.5.2.1.

core document.
& Ear Record Book and specimen is given a unique lab reference
number.
3.5.2.5. Incubate at 35 2 C for 18-24 hours.
Day 2
3.5.3. After incubation (18-24 hours), examine the BA, MAC and other plates, if no
obtained.
request form, microbiology worksheet and follow the instruction in point 9.3
numbers 2 to number 6.
Day 4

3.6.
Flow Chart for Processing of Ear Specimen
Received specimen for Ear Swab

(in transport medium)
BA
MAC
CHOC
SDA
Culture
Incubate 35 2 C
Read media
No growth
Mixed growth
Growth
Identification Test
Sensitivity Test
Report
1. Label
2. Record
3.6.
Work Procedure for Processing of Ear Specimen
Day 1
3.6.2.1.

core document.
& Ear Record Book and specimen unique number is given.
perform.
Day 2
3.6.3. After incubation (18-24 hours),examine the BA, MAC, CHOC and SDA, if no
obtained.
request form and microbiology worksheet.
result in Mycology record book and Eye & Ear record book.
Day 3

3.7. Flow Chart for Processing of Pus and Swab Specimen
Received specimen for Pus

(Stuart/sterile container)
Gram stain (pus only)
Culture
Incubate 35 2 C
1. Label
2. Record
Swab
BA
MAC only
Francis Media
BA (Ano2)
CHOC
SDA
Read media
No growth
Mixed growth
Growth
Identification Test
Sensitivity Test
Report
Pus
only
3.7. Work Procedure for Processing of Pus and Swab Specimen

Day 1
3.7.1. Collect the samples from the specimen receptions
3.7.2.1. If not complete or have any discrepancy of the sample and Request
core document.
in red ink at canter top of the request form.
3.7.2.3. If the sample in acceptable criteria, record the patient data into
Pus/Bone/Tissue/Swab/Tip Record Book and specimen unique
number is given.
3.7.3. Label the culture plate with unique specimen number and a date perform.
Each culture plate is used for two samples.
3.7.4. Inoculate the specimen onto respective culture media.
3.7.5. Aseptically streak the inoculums to produce single colonies.
3.7.6. Incubate all the culture plate at 35 2 C for 18-24 hours
3.7.7. Pus aspirate will be tested for anaerobic bacteria routinely.
3.7.8. Gram stain will be performing only for pus aspirate, refer to Gram stain
procedure.
3.7.9. Record the Gram stain result onto Pus/Bone/Tissue/Swab/Tip Record
Book.
3.7.10. Keep the Gram stain slides into the designated slide box.
Day 2
3.7.11. After incubation (18-24 hours), examine the BA, MAC and other plates, if
no growth, re-incubate and examine after overnight incubation.
3.7.13. Any mixed growth culture more than 2 types of colonies identify according
to colonys morphology. No sensitivity test required. Record the results into
3.7.14. Observe for yellow halo/colouration along the primary and secondary
streak lines of Francis media. If present, alert microbiologist. Give a
preliminary melioidosis and inform to the ward by telephone as soon as
possible after verification (Expected LTAT is 18 hrs from culture).
Day 3
3.7.18. Read the re-incubate plate, if no growth, report as culture no growth was
obtained.
3.7.20. Record all the results into Worksheet Bacteriology, sensitivity form and
Pus/Bone/Tissue/Swab/Tip Record Book completely.
3.7.21. Attach the sensitivity form to the request form
3.7.25. The copy of request form shall be attached with microbiology worksheet
and must be kept in designated place.
3.7.26. Dispatch results that have been verified before lunch and by the end of the
day.
3.8 Flow Chart for Processing of CAPD Fluid Specimen
Grams stain
AFB stain (if indicated)
Received specimen for C

(Bactec bottle)
1. Label
2. Record
Culture
BA
MAC
CHOC
Incubate 35 2 C
Read media
No growth
Mixed growth
Growth
Identification Test
Sensitivity Test
Report
3.8 Work Procedure for Processing of CAPD Fluid Specimen

Day 1
3.8.2.1.

core document.
Blood Record Book and specimen unique number is given.
3.8.2.4. Label the Bactec bottle with unique specimen number.
3.8.2.5. Insert into Bactec 9240.
3.9 Flow Chart for Processing of Catheter Tip Specimen
Received specimen for Tip

(sterile container)
Culture
BA
MAC
Incubate 35 2 C
Read media
Mixed growth
No growth
Growth 15 colonies
Identification Test
Sensitivity Test
Report
1. Label
2. Record
3.9. Work Procedure for Processing of Catheter Tip Specimen

Day 1
3.9.1.
3.9.2.
Collect the samples from the specimen reception.

Check the sample and the request form.
3.9.2.1.

core document.
Pus/Bone/Tissue/Swab/Tip Record Book and specimen unique
number is given.
3.9.2.4. Label the culture plate
3.9.2.5. Dip forceps in 95% alcohol, and flame to sterilize. Allow the
forceps to cool.
3.9.2.6. If the catheter segment is too long to be rolled on the plate, cut it
with the sterile scissors and culture each segment separately.
3.9.2.7. Use sterile forceps to transfer catheter tip from transport container
to BA and Mac agar.
3.9.2.8. Using slight pressure, roll catheter tip back and forth cross the agar
surface at least four times. It is essential that the catheter tip have
good contact with the surface of the plate. (or roll it like a M pattern)
3.9.2.9. If tip is bent and hard to roll, use forceps to pick up tip, and rub all
surfaces onto plate.
3.9.2.10. Dip forceps in 95% alcohol, and flame sterilize.
3.9.2.11. Incubate plate at 35 2 C for 18-24 hours.
Day 2
3.9.3. Examined the culture plate, if no growth, re-incubated overnight.
3.9.4. If there is growth, but less than 15 colonies, it is considered insignificant
growth and no identification and sensitivity test required, report as culture
insignificant growth less than 15 colonies.
3.9.5. If mixed growth (more than 2 types of colonies) identify according to
colonys morphology. No sensitivity test required. Report as mixed growth
with the type of isolated organism.
3.9.6. Identify and perform sensitivity testing on each organism that produces 15
colonies (not more than 2 types of organisms).
3.9.7. Incubate the identification biochemical set and sensitivity at 35 2C for 1824 hours at appropriate atmosphere (CO2 or normal atmosphere).
Day 3
3.9.8. Read the sensitivity plate and biochemical set.
3.9.9. Record all the results into Worksheet Bacteriology sensitivity form and Pus
Record Book completely.
3.9.12. All reports will be validated by Microbiologist before being dispatched
3.9.13. Any discrepancy, refer to the Microbiologist.
3.10.
Flow Chart for Processing of Urine Specimen
Received specimen in sterile

container/boric acid container
Reject
No
Acceptable?
Incomplete request form e.g

no clinical diagnosis or time
specimen collected was not
stated, stamped with TAK
LENGAP in red ink.
Microscopy using KOVA
slide method
Yes
Inoculate onto CLED using uri-strip

for semi-quantitative colony count;
BA and MAC for identification
Incubate for 18-24hrs at 35 2 C
Examine all the plate
Report as culture no growth with cell count

results. Re-incubate for another 24 hrs for
criteria stated at point 9.2.2.2 in the text
No growth
Count the colonies from both impression site of

uri-strip as a total count. Average the count
Growth
Established the colony count and workup the

culture according the criteria describe as in Table
1 and 3 in the text.
A
Report
Reporting of culture results
If only nonuropathogen
isolated, report
as the colony
count (CFU/ml)
normal
urogenital
microbiota
Negative result,
insignificant
results and
mixed growth
with no workup
with correlation
of pyuria follow
instruction in
the Table 4
Issue the report
Growth with
workup, report
each isolates,
colony count
and AST
Mixed growth
mixed growth
and specimen
contaminated,
Repeat c/s if
symptoms persist
3.10.
Work Procedure for Processing of Urine Specimen.
Day 1
3.10.2. Check the sample and the request form:
3.10.2.1. If not complete or have any discrepancy of the sample and request
form, reject the request. The criteria for specimen rejection refer to
core document.
Urine Record Book and specimen unique number is given.
3.10.3. Semi quantitative culture by Uri-Strip methods.
3.10.3.1.
Mix the urine thoroughly.
3.10.3.2. Label the culture plate (BA, Mac & CLED) with unique specimen
number and the date perform. Each culture plate is used for two urine
sample. BA and Mac are used for the isolation of bacteria colonies and
CLED is used for colony count.
3.10.3.3. Dip the angled end or foot of the Uri-Strip quickly into the urine.
3.10.3.4. Drain excess urine by touching the side of the container. Make an
impression of the foot on the well-dried CLED culture media.
3.10.3.5. Each urine sample is done in duplicate side by side.
3.10.3.6. Using a sterile wire loop inoculates the urine onto BA and Mac and
spread out the inoculums to obtain single isolate colonies.
3.10.3.7. Incubate all the culture for 18-24 hours at 35 2 C.
3.10.4. Urine Microscopy
3.10.4.1. Examination of urine microscopy of freshly collected uncentrifuge
urine is perform by using KOVA slide method (Hycor Biomedical
Inc. California,USA) refer to to Product Insert File).
3.10.4.2. The KOVA Glasstic slide 10 with grid chamber is an optical clear,
plastic, and disposable. The slide that holds up to 10 specimens
allowed the user to perform quantitative microscopic counts of pus,
epithelial and red blood cells. Each of the 10 chambers will hold a
standardized 6.6 l of sample and has a depth of 0.1mm.
3.10.4.3. Mix well the urine.
3.10.4.4. Add 6.6 l of uncentrifuge urine to one grid of the KOVA slide.
3.10.4.5. Allow grid to fill by capillary action.
3.10.4.6. Count the pus cell, RBC and epithelial cells under x 400
magnification.
3.10.4.7. If few number of cell seen, use the Low Cell Count Samples
method and for numerous cell count seen, use the High Cell count
Sample
3.10.4.8.
The method is described in the Product Insert File and the value
table is attached in this work instruction.
Day 2
3.10.5. Examine plates after appropriate incubation time (overnight but make a final
reading after 18hrs incubation). CLED culture plate for colony counts, BA
and Mac for further workup such as identification and AST.
3.10.6. Culture with no growth:
3.10.6.1.
Report negative urine culture after 18 -24 hrs incubation as
No Growth.
3.10.6.2.
Re - incubate the culture plate for another 24 hrs if one of
following is true:
a. The suprapubic bladder aspiration or straight catheter method
(in and out catheter).
b. If colonies are too tiny
c. Culture results do not correlate with urine microscopy finding or
clinical conditions ( pus cell >10 cell/l, or symptomatic
patient)
3.10.7. Culture with growth
3.10.7.1.
Examine culture media for the quantity and morphological
type of organisms present.
3.10.7.2.
Count the colonies from the both impression site of UriStrip on CLED and divide by two to get the average. This average
count would be the colony count. Multiply appropriate dilution

factor in CFU/ml
No. of colonies
0
1
1-5
6-24
25
Colony count (CFU/ ml)

0
1000 (103)
1000 -10 000 (103-104)
10 000 100 000 (104 - 105)
100 000 (105)
3.10.7.3.
If growth is mixed, determine of each morphotype of
isolate by examine the BA and MAC.
3.10.7.4.
Workup cultures according to the criteria in Tables 1 and 2
below.
Table 1: Criteria for the identification and susceptibility testing of organisms isolated from
midstream urine collection and indwelling catheter
No. of
isolates
No. of colonies
of each types
CFU/ml of
uropathogens
Work up for
uropathogens
Report
<5
< 104 CFU/ml
No work up
<5
< 104 CFU/ml
ID + AST
5 24
104 CFU/ml
ID + AST
No
significant
growth
< 104
CFU/ml
104
CFU/ml
25
105 CFU/ml
ID + AST
Both 25
105 CFU/ml
ID + AST on
both
105
CFU/ml
105
CFU/ml
Remarks
Pyuria with relevant

clinical information.
Patients on
antimicrobial agents,
female patients with
urethritis, and
symptomatic males or
pyuria ((>10 cell/l)
with diagnosis of
recurrent or chronic
urinary tract infection
and pyuria (>10 cell/ l)
One 25
10 CFU/ml
ID + AST
One < 25
Both < 25
< 105 CFU/ml

< 105 CFU/ml
Ignore
No workup
All uropathogens
< 25
< 105 CFU/ml
No workup
All uropathogens
25
105 CFU/ml
No workup
10
CFU/ml
No
significant
growth
No
significant
growth
Mixed
growth
Table 2: Criteria for the identification and susceptibility testing of organisms isolated from
in and out Catheter (straight catheter) and suprapubic
No. of isolates
No. of
colonies of
each types
CFU/ml of
uropathogens
Work up for
uropathogens
1 uropathogen
with normal
urogenital or
skin microbiota
103 CFU/ml
Minimal ID*
1 uropathogen
Appropriate
dilution factor
ID + AST
2 uropathogen
103 CFU/ml
Minimal ID*
2 uropathogen
Appropriate
dilution factor
3 uropathogen
For each < 5
3 uropathogen
For each 5
Report
Appropriate
factor
dilution
ID + AST on both
isolates
Appropriate
factor
dilution
<104 CFU/m
Minimal ID*
<104 CFU/ml
104 CFU/ml
ID + AST on all
isolates
Appropriate
factor
dilution
* Minimal identification of bacteria is based on morphotype of isolate grows on MAC and BA,
gram stain and minimal testing. The example of minimal testing for Staphylococcus aureus
are catalase positive and coagulase positive; Enterococcus are catalase negative, PYRpositive, nonhemolytic and presumptive Pseudomonas aeruginosa are oxidase positive and
non-lactose fermented.
3.10.7.5. Report any amount of Streptococcus group B in this female age
group (14 60 years old) in pure culture.
3.10.7.6. For identification of the bacteria, refer to work instruction gram
negative and gram positive
3.10.7.7.
Performed AST as indicated in Tables 1 and 2.
3.10.7.8.
List of Uropathogens and Non-Uropathogens as in Table 3
Table 3: List of uropathongens and Non-uropathogen

Uropathogens
Non-Uropathogens (normal skin/urogenital

flora)
Enterobacteriaceae
Viridans group streptococci
Pseudomonas aeruginosa
Lactobacillus
Other gram negative bacilli
Diphtheroids
Entetococcus spp.
Bacillus spp.
Beta-hemolytic Streptococcus
Staphylococcus spp.
Corynebacterium urealyticum
Uropathogens
Non-Uropathogens (normal skin/urogenital

flora)
Staphylococcus aureus
Staphylococcus saprophyticus (female 12 -60
years old only)
Others Staphylococcus coagulase negative*
Aerococcus urinae*
*consider as uropathogen only when present in amounts > 10 folds of others non uropathogens
Day 3
3.10.7.9. Examined and read the Biochemical identification media and
sensitivity plate.
3.10.7.10. For incomplete identification, complete the additional test and
inform microbiologist every day of the progress.
3.10.7.11. Issue identification and AST results
3.11.Reporting results
3.11.1. Report urine microscopy performed by KOVA slide method.
3.11.2. If no growth is observed on all media, report as Culture No Growth.
3.11.3. If only urogenital or skin microbiota is observed, report as such.
Example 100,000 CFU/ml normal urogenital microbiota.
3.11.4. Reporting of the negative result, insignificant results and mixed

growth with no workup with correlation of pyuria are describe in the
table 4 below:
Table 4: The Description pyuria, colony count and reporting of urine culture.
No
.
1.
Pus cell
(KOVA)
< 10/ l
Colony count
(CLED)
0
Description
Report
Culture No
growth
2.
> 10/l
Culture No
growth
3.
> 10/l
1-5 colonies
Colony count
1000-10,000
organisms/ml
urine
Urine Microscopy Pus cells __/l ,RBC

___/l Epithelial cell ____/l
Culture; No growth
Repeat c/s if symptoms persist
Urine Microscopy Pus cells __/l ,RBC
___/l Epithelial cell ____/l
Culture; No growth
UTI may be present
Repeat c/s if symptoms persist
Urine Microscopy Pus cells ___/l ,
RBC ___/l, Epithelial cell ____/l
Insignificant growth
<104
UTI may be present
Rpt c/s if symptoms persist
4.
> 10/l
< 25 colonies
Mixed growth
2 types
5.
> 10/l
> 25 colonies
mixed growth
3 types
Insignificant
bacteriuria
<100 000
organisms/ml
urine
Borderline
Significant
Colony count
> 100 000
organisms / ml
Heavily mixed
growth

Borderline Significant growth
104- 105 CFU/ml
UTI probable
UTI Probable
However specimen contaminated
3.11.5. Mixed culture as specified in table 1 are reported as mixed growth and
specimen contaminated, Repeat c/s if symptoms persist.
3.11.6. Growth with workup as in Tables 1 and 2 report the organisms name, AST
and corresponding colony count (CFU/ml).
3.11.7. Inform the ward of unusual isolate such as Salmonella typhi or Burkholderia
pseudomallei.
3.11.8. Record all the results into Worksheet Bacteriology (PAT/MICRO/BAC/02)
sensitivity form (PAT/MICRO/BAC/01) and Urine Record Book completely.
3.11.9. Attach the sensitivity form to the request form (PER-PAT 301).
3.11.11. All reports will be validated by microbiologist before being dispatched.
3.11. Flow Chart for Processing of Stool Specimen
Received specimen for Stool (in

sterile cup)
BA
MAC
CHOC
SDA
Culture
Campy
Incubate 35 2 C
Read media
No growth
Mixed growth
Growth
Identification Test
Sensitivity Test
Report
1. Label
2. Record
3.12.
Work Procedure for Processing of Stool Specimen
Day 1
3.12.2.1. If not complete or have any discrepancy of the sample and request
core document.
Stool Record Book and specimen unique number is given.
perform.
Day 2
3.12.3. After incubation (18-24 hours) examine the BA, MAC, CHOC, SDA and
Campy if no growth, re-incubate and examine after overnight incubation.
obtained.
result in Mycology record book and sSool record book.
3.12.10. Incubate the respective biochemical identification media/kit and
sensitivity media in appropriate condition.
Day 3
3.12.12. Record all the results into microbiology worksheet, sensitivity form, and
Stool Record Book completely.
4.0. References
5.0. Appendices

Workflow Bactriology

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Contents

Specimen collection, transport & processing

2.0 General Workflow - Microbiology Laboratory

2.0 General Work Procedure Microbiology Laboratory

3.0 Specimen collection, transport & processing

Received specimen for anaerobic

Anaerobic Blood agar

Work Procedure for Identification of Anaerobic Bacteria

If not complete or have any discrepancy of the sample and

Observed for any growth.If no growth. Report as No

Record morphology of each colony type, such as pitting,

3.2 Flow Chart for Processing of CSF Specimen

Received specimen for CSF

Incubate 35C (2)

Work Procedure for Processing of CSF Specimen

INITIAL PROCESSING OF SPECIMEN

b. Count number of both cells according to KOVA insert (Low

e. Cell count not performs for bloody sample and report as

3.2.6. LATEX AGGLUTINATION

Aseptically streak the drop on the culture media to produce

3.3 Flow Chart for Processing of Bone & Tissue Specimen

Received specimen in sterile

Add sterile tryptone water into

3.3 Work Procedure for Processing of Bone & Tissue Specimen

All specimens are cultured into anaerobic blood agar.

Ziehl-Neelson stain is done if requested/clinically indicated suspicion of

3.3.10 Give specimen to TB Laboratory to culture onto Lowen-Jensen media.

Flow Chart for Processing of Eye Specimen

Received specimen for Eye Swab

3.4. Work Procedure for Processing of Eye Specimen

If not complete or have any discrepancy of the sample and request

3.5 Flow Chart for Processing of Contact Lens Specimen

Received specimen for Contact

3.5 Work Procedure for Processing of Contact Lens Specimen

If not complete or have any discrepancy of the sample and request

3.5.12. Place all completed reports into respective drawer.

Flow Chart for Processing of Ear Specimen

Received specimen for Ear Swab

Work Procedure for Processing of Ear Specimen

If not complete or have any discrepancy of the sample and request

3.6.11. Read the Biochemical identification media/kit and sensitivity plate.

3.7. Flow Chart for Processing of Pus and Swab Specimen

Received specimen for Pus

Gram stain (pus only)

3.7. Work Procedure for Processing of Pus and Swab Specimen

3.8 Flow Chart for Processing of CAPD Fluid Specimen

Received specimen for C

3.8 Work Procedure for Processing of CAPD Fluid Specimen

If not complete or have any discrepancy of the sample and request

3.9 Flow Chart for Processing of Catheter Tip Specimen

Received specimen for Tip

3.9. Work Procedure for Processing of Catheter Tip Specimen

Collect the samples from the specimen reception.

If not complete or have any discrepancy of the sample and request

Flow Chart for Processing of Urine Specimen

Received specimen in sterile

Incomplete request form e.g

Inoculate onto CLED using uri-strip

Report as culture no growth with cell count

Count the colonies from both impression site of

Established the colony count and workup the

Reporting of culture results

Issue the report