QualityControl Reagents Tests

CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURES
Quality Control of Reagents and Tests – SOP No: CRM-SOP 21
CAREC PAHO WHO EUROPEAN UNION
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN

A CARIFORUM Project Funded by the European Union and Implemented by CAREC
Quality Control of Reagents and Tests
Title: Quality Control of Reagents and Tests SOP Number: CRM-SOP: 21
Version: 1
Page Number: 1 of 88
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007
Methods Drafting Group Review Date: 1st September 2008
TABLE OF CONTENTS
Acknowledgements 2
Introduction 4
Amendment procedure 5
1.0 Reagent and Test Quality Control 6
2.0 Process Flow Chart For Quality Control of Bacteriology Tests 10
3.0 Record Chart for Quality Control of Reagents and Stains 11
4.0 Bacitracin Differentiation Test 12
5.0 Beta-Lactamase (Cefinase) Test 16
6.0 Bile Solubility Test 20
7.0 Catalase Test 25
8.0 Camp Test 32
9.0 Coagulase Test 36
10.0 Clindamycin resistance in Staphylococcus species and

Beta-hemolytic Streptococcus species 42
11.0 Germ Tube Test 44
12.0 India Ink Test 50
13.0 Indole Test 54
14.0 Novobiocin Susceptibility Test 60
15.0 Optochin Test 64
16.0 Oxidase test 68
17.0 PYR Test 72
18.0 Urease test 76
19.0 X and V test 80
20.0 Ziehl–Neelsen Stain Test 84

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Acknowledgements
The ‘Strengthening of Medical Laboratory Services in the Caribbean’ (SMLS) Project, would like to thank
the following people for their participation in researching and writing the microbiology standard
methods, and also the individual countries for supporting this initiative.
Many thanks also for the facilitators of the standard methods meetings, the method editors and
administrative support.
Participants
Country Name Organisation

Aruba Ms. Astrid Dirksz Landslaboratorium
Anguilla Ms. Everette Duncan Princess Alexandra Hospital, Stoney Ground
Bahamas Mr. Allison Scavella Princess Margaret Hospital
Barbados Ms. Gail Trotman Public Health Laboratory
Ms. Juliana Applewaite Queen Elizabeth Hospital
Mr. Edmund Blades Public Health Laboratory
Belize Ms. Solitaire Parra Maza Central Medical Laboratory
British Virgin Islands Ms. Allene Brewley Peebles Hospital
Ms. June Greene Medicure Ltd.
Ms. Wayveney Armstrong MEDICAL Diagnostic Laboratory
Cayman Islands Mr. Dale Andrew Chin Caymans Islands Health Services Authority
Curacao Mr. Osric Wanga Analytical Diagnostic Centre
Ms. Helga Leito Analytical Diagnostic Centre
Grenada Ms. Sonia Ann Edwards St Georges Hospital
Guyana Ms. Alexis Wilson Pearson Georgetown Public Hospital
Ms. Ede Tyrell Langevine University of Guyana
Jamaica Ms. Rayaad Khan Central Medical Laboratories Ltd
Ms. Valerie Levy UWI Microbiology Department
Ms. Heather Wint Caledonia Medical Laboratory (Biomedical)
Ms. Adriene Kellier Cornwall Regional Hospital
Mr. Norman S. Burke National Public Health Laboratory
St Lucia Mr. Martin S. Mc Kenzie Ezra Long Laboratory
St Vincent and Mr. Elliot Samuel Milton Cato Memorial Hospital
the Grenadines
Caribbean Epidemiology Ms. Radha Gosein CAREC
Centre (CAREC) Ms. Michele Nurse-Lucas CAREC
Ms. Denise Clarke CAREC
Dr. Ashok Rattan CAREC
Trinidad Ms. Zobida Khan Mohammed Trinidad Public Health Laboratory (TPHL)
Mr. Anthony Bayley Trinidad Public Health Laboratory (TPHL)
Mr. Jawaheerlal Mewahlal College Of Science, Technology And Applied
Arts of Trinidad and Tobago (COSTAATT)
Ms. Monica Pollard Port of Spain General Hospital
Dr. William Swanston Eric Williams Sciences Medical Complex
Turks and Caicos Islands Ms. Lessonjule Lyons Doney Grand Turk General Hospital Laboratory
Ms. Peggy Hermitt Myrtle Rigby Hospital

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Facilitators/Editors
Country Name Organisation

Canada Dr Harold Richardson Quality Management Program –
Laboratory Section
Trinidad Ms. Julie Sims Strengthening of Medical Laboratory Services
in the Caribbean (SMLS)
United Kingdom Ms. Jacki Watts Bristol Health Protection Agency
United Kingdom Ms. Valerie Bevan Health Protection Agency (HPA UK)*
Administrative Support
Function Name Organisation

Administrative Support Ms. Stacey-Mae Stephens SMLS Project
Administrative Support Ms. Reesa Moonsie SMLS Project
Rapporteur Ms. Margaret Hunte Independent
Graphic Design and Support
Function Name Country

Graphic Design Ms. Cathleen Jones Trinidad & Tobago
Graphic Support Mr. Adrian Nicholls United Kingdom
Graphic Support Ms. Karen Lara-Augustine Trinidad & Tobago
*The Health Protection Agency, England, is acknowledged for the knowledge and expertise of their staff and working
groups in the UK who develop National Standard Methods, on which the Caribbean Regional Microbiology Standard
Operating Procedures are based.

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INTRODUCTION
The Caribbean Regional Standard Methods include a selection will be based on a review of EQA results, most
variety of standard, validated methods, produced as common and/or critical tests. Part of the method
a single standard operating procedure (SOP) for use in a standardization process will be an ongoing review and
variety of levels of microbiology laboratory service. amendment procedure. The CSMDG consists of
It is intended that these methods provide detailed microbiology laboratory representatives from most of
instructions for microbiology services for microbiological the CARIFORUM countries, all of whom were nominated
investigations, in order to provide accurate, reliable and to the task by the CRMC.
reproducible results which will have clinical utility. These
methods may be adopted by laboratories within the This initiative should enable the region to implement a
region, or adapted, provided that such adaptations use standardized and constructive method for ensuring that
an evidence-based validation process. validated methods are available for the region, and that
they are updated as required.
These methods have been developed by the Caribbean
Regional Microbiology Standard Methods Drafting Advantages of using regionally validated methods are
Group (CSMDG) in response to a request by the to improve quality, make better use of resources,
Caribbean Regional Microbiology Council (CRMC), reduce costs, enable central procurement & media
which was set up by the CARIFORUM Project entitled preparation, facilitate staff training and transfers due to
‘Strengthening of Medical Laboratories in the horizontal integration, a reduction in variability of service
Caribbean’ to strengthen specifically the microbiology provision, an improved quality of surveillance data,
services in the Caribbean Region. The Project was and the purchase of appropriate equipment. A major
initiated in response to findings which indicated that advantage is that the availability of regional standard
there was an unacceptable level of error in laboratories methods would assist microbiology laboratories with
within the region. External quality assessment results documentation for accreditation
revealed that microbiology laboratories were not
performing well and feedback from the region via Although the CSMDG has taken every care with the
laboratory staff, lab managers and directors was that preparation and issue of these standard procedures,
they felt that guidance in microbiology requirements was and they have been validated regionally, nationally and
required. internationally, the CSMDG, or any other organization,
cannot be responsible for the accuracy of any statement
The background for this initiative is a worldwide move to or representation made or the consequences arising
implement standards in all areas, which has now from the use of or alteration to any information
extended to include medical laboratories. As tourism is contained in them. These procedures are intended
so vital to the region’s economy, the need for accurate solely as a resource for practicing microbiology
diagnosis and treatment is paramount. It was accepted professionals in the field, operating in the Caribbean
that there is a requirement for validated methods for region, and specialist advice should be obtained where
accreditation purposes and providing validated standard necessary. If changes are made to the original
methods will assist in the move towards accreditation. publication, it must be made clear where changes have
been made to the original document. When referring to
The methods will be chosen for standardization by the these SOPs in successive documentation, the CSMDG
Caribbean Regional Microbiology Council, and this should be acknowledged.

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Amendment procedure
Controlled Document Reference CRM-SOP 21
Controlled Document Title Standard Operating Procedure for Quality Control of

Reagents and Tests
Each Regional Standard Method should be reviewed annually by the Caribbean Standard Methods Drafting
Group. Any amendments should be validated and authorized by an agreed process, and referenced.
Each Regional Standard Method has an individual record of amendments. The current amendments are listed
on this page.
On issue of revised or new pages, each controlled document should be updated by the copyholder in the
laboratory.
Amendment Issue Number Insert Issue Page Section(s) Amendment

Number/Date Discarded Number involved

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1.0 Reagent and Test Quality Control
1.1 Test and Reagent Quality Control Procedures
1.2 Purpose
To ensure that correct validated procedures are followed when performing quality control (QC) of reagents and
tests.
1.3 Introduction
All tests performed in the microbiology laboratory must have quality control performed in order to verify and
validate test results. Quality control results provide evidence that the procedures were followed as intended
and allow the user to report results with confidence. This standard operating procedure is a compilation of
some of the most common test and stains performed in the clinical laboratory. Each procedure is accompanied
by relevant flowcharts and results record charts.
1.4 Scope
The procedure provides detailed instructions for microbiology services as offered by regional laboratories. It
may be adopted or adapted by any laboratory as needed, provided that such adaptations use an evidence-
based validation process.
1.5 Staff Competency Requirements

Laboratory personnel, trained and assessed to be competent to perform this procedure
1.6 Safety Instructions

Refer to CRM-SOP 20: Safety in the Microbiology Department
Level 2 containment; requires all personal protective equipment (PPE). Goggles should be used when
performing processes that may give rise to aerosols
All microbiological samples for processing should be considered a potential source of transmissible infections
so universal safety precautions should be observed.
Hands should be thoroughly washed with soap and water before and after handling all specimens.
Disinfect all work surfaces with 70% alcohol or a freshly prepared 10% bleach solution prior to testing and after
processing
All samples and reagents should be properly discarded according to the current standards for disposal of
hazardous waste.
Samples and culture plates should be autoclaved before finally discarding.
Any procedure which is likely to produce aerosols should be performed in a biosafety cabinet.

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1.7 Pre-Examination Procedure

1.7.1 Sample Type
Usually isolates for identification, presented on agar plate media
1.7.2 Sample Collection

N/A
1.7.3 Transport & Storage

Refer to original SOPs.
1.7.4 Rejection criteria

Mixed cultures
Old cultures
Dessicated cultures
1.7.5 Relevant clinical information

N/A
1.8 Media, Reagents and Equipment

See individual SOPs.
1.9 Examination Procedures

a. Perform positive and negative QC at the same time as the test.
b. If both controls pass (i.e. positive controls are positive and negative controls are negative) continue
processing.
c. If either or both QCs fail then corrective actions must be taken. These should include a review of procedures
as follows (this is not an exhaustive list):
• Are SOP’s for the procedure in place.
• Are SOP’s for the procedure current.
• Is an audit trail for the procedure available and completed.
• Was the correct procedure followed.
• Were correct quantities of reagent used.
• Were correct timings followed.
• Was colour recognition correct.
• Were correct drying/fixing protocols followed.
• Were temperatures and pH of water monitored and were they correct.
• Was slide/equipment contaminated.
• Were staff performing the procedure trained and competent to do so
• Were reagents and media within their expiry date.
• Were incubator/refrigerator/waterbath/freezer temperatures recorded and were they correct.
• Check reagent storage conditions and usage.
• Were storage temperatures appropriate during transporting.
• Were reagents allowed to stabilize at recommended temperatures prior to use?
• Were reagents used in their intended capacity and recommended concentration.
• Were correct QC organisms used for QC.
• Were QC organisms propogated and stored correctly

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1.9.1 Quality Control

Perform appropriate QC protocol for individual staining. Use standard bacterial strains for checking validity of
media and identification tests. Quality Control should be run concurrently with tests
Refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

Refer to CRM-SOP 18: Preparation and Quality Control of Media
Standard reference control strains should be used wherever possible
Results of all QC testing should be recorded on the appropriate QC forms available in the department, and
which can be photocopied from this CRM-SOP
Results of all tests are invalid if the QC test results are not as expected
Ensure that reagents, stains and media are not used beyond the expiry date
Check all reagents and stains before use to ensure that they are free from contamination, debris or deposits.
All media used should be examined visually just before use to ensure that there is no contamination or
deterioration appearing as lysis, discolouration, drying, shrinkage, or cracking of the media.
Refer to departmental SOPs for further details.
1.9.2 Microscopy
Gram films should be performed to validate Gram stain and morphology, and presumptive identification of
organism, before more extensive and costly test are performed.
Refer to CRM-SOP 7: Gram Stain for methodology
1.9.3 Culture
Occasionally sub-culture, or culture to obtain discrete colonies may be required.
Refer to CRM-SOP 22: Inoculation of Culture Media
1.9.4 Identification
Gram films should be performed to validate Gram stain and morphology, and presumptive identification of
organism, before more extensive and costly test are performed.
Refer to CRM-SOP 7: Gram Stain for methodology
1.9.5 Susceptibility testing

N/A
1.9.6 Referral
Organisms may be referred for confirmation of identification, or identification of more esoteric organisms, or
confirmation of serotype.

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1.10 Post-Examination Procedures

1.10.1 Reporting
If QC passed: Report test results and record QC results.
If QC failed: Take corrective action and repeat entire test procedure until QC passes prior to sending out results.
1.10.2 Sample Retention, Storage and Disposal

All plates should be retained until 48 hours after the clinician receives the results. Representative plates should
be stored in a monitored refrigerator for retrieval
1.11 Limitations and Pitfalls

1) A contaminated batch of reagents may be used, leading to incorrect test and QC results
2) QC organisms may lose their QC factors if not propogated and stored correctly, or are sub-cultured too
often, leading to incorrect QC results.
3) Test/QC cultures may be too old to yield intended expected quality control results.
1.12 References
1. Health protection Agency BSOP TP 36.
2. Monica Cheesbrough, Medical Laboratory Manual for Tropical Countries, Volume II, Microbiology. 1984.
Butterworth-Heinemam, Microbiology Edition, EL/BS with Tropical Health Technology, 1991, p60–61
3. Ridley M.J., Ridley D.S. Stain techniques and morphology of mycobacterium lepta – leprosy
4. Jokahaski S. Handbook of direct smear examination of sputum for tubercle bacillus, SE AMIC publication,
No. 4, 1975.
5. I Collins CH, Grange JM, Yates MD. Tuberculosis Bacteriology 2nd Edition, 1997.
6. Isenberg, Henry D, Essential Procedures for Clinical Microbiology ASM Press 1998, pp 176–178.
7. Murray, PR et al; Manual of Clinical Microbiology, 7th ed., 1999 pp 1678.

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2.0 Process Flow Chart For Quality Control of Bacteriology Tests
Perform QC concurrently with test
Has the QC passed?
Yes Investigate all

No factors that may
lead to a discrepancy.
Document and report

patient results and
record QC results.
Are all OK?
No Yes
Take corrective action

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3.0 Record Chart for Quality Control of Reagents and Stains
Tests/Stains Reagents Quality Control ATCC # Observed Results Interpretation of

Used Organisms Results
Gram Stain
ZN stain
India Ink
Catalase Test
Germ Tube Test
Indole Test
Oxidase Test
Camp Test
Novobiocin Test
Optachin Test
Bile Solubility Test
Beta-Lactamase
Test
Bacitracin
Differential Test
PYR Test
X and V
Factor Test
Comments: ............................................................................................................................................................
Reports reviewed by: .............................................................................. Date:....................................................

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4.0 Bacitracin Differentiation Test

4.1 Purpose
To ensure the correct validated procedure is followed for performance of the Bacitracin Differentiation Test. To
produce accurate, reliable, reproducible results having clinical utility.
4.2 Introduction
This test is used for the presumptive identification of Group A Streptococcus (Streptococcus pyogenes) and
for differentiation from other ß-hemolytic Streptococcus species.
Most strains of Group A Streptococcus (GAS) are susceptible to 0.04μg bacitracin disc. However, test organism
resistant bacitracin should be tested further using a grouping method to confirm identification

Refer to CRM-SOP 21: Quality Control of Reagents and Tests, Section 2

• 0.04μg Bacitracin Differentiation Discs (Difco/Oxoid)
• Blood agar (BA)
• Culture loop/ sterile cotton swab
• Forceps
• 37ºC Incubator
4.5 Quality Control

Quality Control should be run concurrently with tests
Refer to CRM-SOP 21: Quality Control of Reagents and Tests: Section 2
4.5.1 Quality Control Organisms

Positive Control: Streptococcus pyogenes ATCC 19615
Negative Control: Streptococcus agalactiae ATCC 13813
4.6 Examination Procedure

• Inoculate surface of blood agar with the suspect ß-hemolytic streptococci using sterile loop or cotton swab
• Using aseptic technique use forceps to place disc onto inoculated surface
• Incubate aerobically 35 –37ºC for 18–24 hours
4.7 Interpretation & Reporting

• Check blood agar for zone of inhibition
• Susceptible – any zone of inhibition – Presumptive Group A Strep
• Resistant – no zone of inhibition.
4.8 Limitations and Pitfalls of the Procedure

Other ß-Hemolytic Streptococci may be susceptible to bacitracin. Therefore this test should only be used for
presumptive identification and should be followed by a confirmation test e.g. Lancefield Grouping or PYR test.

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4.9 References
1. Difco Differentiation Disks Bacitracin package insert
2. Murray, PR, et al, Manual of Clinical Microbiology, 7th Edition 1999, pp 287.

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4.11 Procedure flowchart for Bacitracin Differentiation Test
Inoculate suspect colonies

to a blood agar plate.
Add a bacitracin disc to

the surface aseptically

and repeat test along
with controls
Incubate aerobically at
35 –37ºC for 18 – 24 h
Examine plate for

zone of inhibition
Is QC OK? No
Yes
Is there any zone of inhibition?
Yes No
Presumptively Group A May not be Group A

Streptococcus Streptococcus
Do confirmation test for

Group A Strep e.g. Lancefield
Grouping/PYR test.

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4.12 Bacitracin Control Results
Date Batch Positive Control Negative Control Comments Sign

pyogenes agalactiae
ATCC 19615 ATCC 13813

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5.0 Beta-Lactamase (Cefinase) Test

5.1 Purpose
To detect the production of Beta-lactamase in organisms resistant to penicillin and cephalosporin.
5.2 Introduction
The Cefinase disc is impregnated with the chromogenic cephalosporin, nitrocefin. This compound exhibits a
very rapid colour change from yellow to red as the amide bond in the lactam ring is hydrolyzed to lactamase.
When a bacterium produces this enzyme in significant quantities, the yellow-coloured disc turns red in the area
where the isolate was smeared. Although other penicillins and cephalosporins may be used as substrates for
specific enzymes, nitrocefin has the wide spectrum of susceptibility and sensitivity of the commercially
available lactams. It is not known to react with other microbial enzymes.


• Cefinase disc
• Sterile loop or applicator stick
• Sterile distilled water
• Empty sterile Petri dish or microscope slide
• Single disc dispenser or forceps
5.5 Quality Control



Control reference cultures should be run with each group of unknowns. The following organisms are
recommended for use as control strains.
Control Strains Expected Results
Staphylococcus aureus ATCC 29213 Positive
Haemophilus influenzae ATCC 10211 Negative

1) Use a single disk dispenser or forceps and dispense the number of required discs from the cartridge into an
empty Petri dish or onto a clean microscope slide.
2) Moisten each disc with sterile distilled water (Note: a positive and negative control should be part of each
run).
3) With a sterile loop or applicator stick remove several well isolated colonies and smear onto disc surface.
4) Observe for a colour change in the appropriate time.

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5.7 Interpretation and Reporting

A positive reaction is a yellow to red colour change on the area where the culture was applied.
Note: Colour change does not usually develop over the entire disc. A negative result will show no colour
change on the disc. For most bacterial strains a positive result will develop within 5 minutes. However reactions
for some staphylococci may take up to 1 hour.
Reaction
Organism Results Interpretation
Time
Staphylococcus aureus Positive 1 hr. Resistant to penicillin,
ampicillin, carbenicillin, and ticarcillin.
Probably susceptible to cephalothin,
methicillin,
oxacillin, naficillin and other penicilli-
nase-resistant penicillins.
Haemophilus influenzae Positive 1 min. Resistant to ampicillin. Susceptible to
cephalosporins.
Neisseria gonorrhoea and Moraxella Positive 5 min. Resistant to penicillin.
catarrhalis
Enterococcus faecalis Positive 5 min. Resistant to penicillin and
ampicillin.
Anaerobic bacteria Positive 30 min. Probable identification is Bacteroides
species. Probably resistant to penicillin
and may be resistant to
cephalosporins including cefotaxime
and rarely cefoxitin.
Susceptibility should be confirmed by growth-dependent susceptibility tests. Negative results imply but do not
guarantee susceptibility. If ATCC quality controls fail to produce the desired results test results must not be
reported. The expiration dates, QC strains, Purity and freshness of colonies and times for reading test results
should be checked and test should be repeated.
5.8 Limitations
As many strains produce inducible Beta–lactamases, it is imperative to take the colony for testing from around
the disc of a Beta–lactam antibiotic.
The efficacy of this test in predicting the lactams resistance of microorganisms other than Neisseria
gonorrhoea, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus, Enterococcus and certain
anaerobic bacteria is unproven. Resistance to lactams antibiotics has been rarely reported in some of the
above organisms without the production of Beta-lactamase. In these cases, resistance mechanisms such as
permeability barrier have been postulated. There, the lactamase test should be used as a rapid supplement
and not a replacement for conventional susceptibility testing. For some strains of staphylococci, particularly
S. epidermidis, an inducible lactamase has been described, that might result in a false negative lactamase
reaction, with a strain which is resistant to penicillin and ampicillin.
5.9 References
1. Marray P.A., et al. Manual of Clinical Microbiology 7th ed.1999
2. Technical Manual Beta-lactamase (Cefinase Test) Toronto Medical Laboratories/Mount Sinai Hospital
Microbiology Department: revised November 2002.

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5.10 Procedure flowchart for Beta-Lactamase (Cefinase) Test
Dispense nitrocefin discs
Moisten discs with sterile

distilled water
Using an applicator stick or sterile

loop smear well-isolated colonies
onto disc surface
Check for colour

change within the
appropriate time
Investigate
Does the QC pass? and take
Yes No
corrective
action
Document and report results

and record QC results

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5.11 Beta-lactamase Control Results

Staphylococcus Haemophilus
aureus influenzae
ATCC 29213 ATCC 10211
Comments: ..................................................................................................................................
Reviewed and dated by: ..............................................................................................................

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6.0 Bile Solubility Test

6.1 Purpose
To ensure the correct, validated procedure is followed for processing the bile solubility test to provide accurate,
reliable, reproducible results having clinical utility.
6.2 Introduction
This bile-insoluble test is used specifically to differentiate between Streptococcus pneumoniae (bile soluble)
and other bile-insoluble Beta-haemolytic streptococci (not bile soluble).
The bile solubility test is used to determine the ability of bacterial cells to lyse in the presence of bile salts, within a
specified time. S. pneumoniae possesses an autolytic enzyme which lyses the cell’s own wall during division. The
addition of bile salts (sodium deoxycholate) activates the autolytic enzyme and the organisms rapidly autolyse.
Other α-haemolytic streptococci do not possess such an active system and therefore do not dissolve in bile.
A heavy inoculum of the test organism is emulsified in physiological saline to give a turbid suspension. The bile
salt sodium deoxycholate is then added. The test can also be performed by adding the bile salt to a broth
culture of the organism.
The bile salt dissolves S. pneumoniae by a clearing of the turbidity within 10 –15 minutes. Viridans streptococci
are not dissolved and therefore there is no clearing of the turbidity.


• Method 1–2% solution of sodium deoxycholate in water. Adjust pH to 7.0.
• Method 2–10% solution of sodium deoxycholate in water. Adjust pH to 7.0.
• 0.85% solution of sodium chloride in water.
• Bacteriological straight wire/loop (preferably nichrome) or disposable alternative.
• Quality control organisms.
6.5 Quality Control


6.5.1 Quality controls organisms

Positive control: Streptococcus pneumoniae ATCC 6305
Negative control: Enterococcus faecalis ATCC 29212

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6.6.1 Method 1 (Plate)
• This method only works on large or mucoid colonies, otherwise results may be subjective
• Select a well-isolated single colony from a blood or chocolate agar plate. Circle the colony on the bottom of
the Petri dish. This will help locate it after testing. Place one drop of 2% sodium deoxycholate directly on the
colony. Incubate at 37ºC for up to 30 minutes. Do not invert the plate. The lid may be left slightly open to aid
evaporation.
• When the reagent has dried, examine the area for lysis or disintegration of the original colony
6.6.2 Method 2 (Tube)

• Prepare a heavy suspension of a pure culture in 1.0ml of 0.85 saline.
• Divide the suspension into two tubes (one test and one control).
• Add 0.5ml of 10% sodium deoxycholate to the test suspension and 0.5ml of 0.85% saline to the control.
• Gently mix both suspensions and incubate at 37ºC for up to 15 minutes.
• Examine for evidence of clearing of turbidity in the tube marked ‘test’ compared with the saline control.

6.7.1 Method 1 (Plate)
Positive result: colony lysed or disintegrated
Negative result: no change
6.7.2 Method 2 (Tube)

Positive result: suspension clears in tube labeled ‘test’ and probably S. pneumoniae remains turbid in control
Negative result: suspension remains turbid in both tubes and organism is probably not S. pneumoniae.
There should be no clearing of turbidity in the tube to which 0.85 % saline was added. If there is, repeat the test.
6.7.3 Report
Bile solubility test positive:S. pneumoniae
Bile solubility test negative: Organism is probably not S. pneumoniae

• The test should not be performed on old cultures, as the active enzyme may be lost.
• Some strains of S. pneumoniae are not dissolved by bile salts.
• Very occasionally some strains of viridans streptococci give a positive result.
6.9 References
1. Health Protection Agency: BSOP TP 5
2. Chesbrough M., Medical Laboratory Manual for Tropical Countries: Volume II: Microbiology,..ed., EL/BS with
3. Tropical Health Technology/Butterworth-Heinemann, 1991, p.59–61

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6.10 Procedure Flowchart for Bile Solubility Test – Method 1 (Plate)
Select a well-isolated
single colony from a blood or
chocolate agar plate
Place one drop of 2% sodium

desoxycholate directly on the suspected
colony and QC control colonies
Incubate at 37ºC for

up to 30 minutes
Are the QC results

Yes No
as expected?'
Did suspected colony

lyse or disintegrate?
Yes No
Positive result Negative result
Organism is probably not

Probably Streptococcus pneumoniae
Streptococcus pneumoniae

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6.11 Procedure Flowchart for the Bile Solubility Test Method 2 (Tube)
Prepare a heavy suspension of a

pure culture and QC in 1.0ml of
0.85% saline
Divide the suspension into two tubes: one test and one control
Tube No. 1: Test tube Tube No. 2: Control tube
Add 0.5ml of 10% sodium desoxycholate to the

Add 0.5ml of 0.85% saline to control tube.
test tube, gently mix and incubate at 37°C for 15
Gently mix and incubate at 37°C for 15 minutes
minutes
Examine for evidence of clearing of turbidity in

test tube compared to control tube
No Take corrective
Did controls pass?
action
Yes
Suspension clears in test

Suspension remains turbid
tube and remains turbid in
in both tubes
control
Positive result-probably
Negative result-probably not
Document test and QC results and report results

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6.12 Bile Soubility Control Results

Streptococcus Enterococcus
pneumoniae faecalis
ATCC 6305 ATCC 29212
Comments: ..................................................................................................................................

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7.0 Catalase Test

7.1 Purpose
To ensure the correct validated procedure is followed for processing the catalase test, to produce accurate,
7.2 Introduction
The catalase test is particularly useful for distinguishing between Staphylococcus species which are catalase
positive, and Streptococcus species which are catalase negative. The test is to detect the catalase enzyme present
in most cytochrome-containing aerobic and facultative anaerobic bacteria. Streptococcus and Enterococcus
species are exceptions.
The catalase test is used to detect the presence of catalase enzymes by the decomposition of hydrogen
peroxide to release oxygen and water. Hydrogen peroxide is formed by some bacteria as an oxidative end-
product of the aerobic breakdown of sugars and if allowed to accumulate is highly toxic. Catalase either
decomposes hydrogen peroxide or oxidizes secondary substrates.The addition of 1% glucose reduces
Pseudomonas-catalase reactions. Catalase acts as a catalyst in the breakdown of hydrogen peroxide to
oxygen and water.
An organism is tested for catalase production by bringing it into contact with hydrogen peroxide. If a rapid
liberation of bubbles of oxygen is present, the gaseous product of the enzyme activity is observed and this
indicates a positive test result, the organism is a catalase producer. A negative result is noted when no bubbles
are formed.

• Catalase testing of bacteria can be hazardous due to the release of bacteria-laden aerosols liberated oxygen.
• Hydrogen peroxide is an irritant.
Reagents Materials Equipment
3–5% hydrogen peroxide Test organisms from chocolate agar Bacti-cinerator

are recommended.
Clean glass slides

Clean capped test tubes
(plastic or glass) or Bijoux
bottles
Bacterial straight nichrome wire or

nichrome loop or disposable loop

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7.5 Quality Control



Quality Control should be performed daily at the same time as test samples.
Positive control: Staphylococcus aureus ATCC 25923


7.6.1 Tube or Bottle Method
• Place approximately 0.2 ml of hydrogen peroxide solution in a test tube or bijoux bottle.
• Carefully pick a colony to be tested with (disposable) loop.
• Rub the colony onto the inside wall of the bottle above the surface of the hydrogen peroxide solution.
• Cap the tube or bottle and lift it to allow the hydrogen peroxide solution to cover the colonies.
• Look for vigorous bubbling occurring within 10 seconds.
7.6.2 Agar Slant Method

• Add 3–4 drops of hydrogen peroxide to an overnight growth on an agar slant and replace the cap.
7.6.3 Cover-Slip Method

• One drop of hydrogen peroxide solution is placed on a slide.
• A small portion of the suspect colonies is placed on the center of a coverslip.
• Invert the coverslip and place in on a drop of hydrogen peroxide solution.
• Use a hand lens if necessary to see very slight catalase production.

Positive Results: Vigorous bubbling indicates the presence of catalase
Negative Results: No bubbling
7.7.1 Report
Catalase Positive or Catalase Negative

• Control organisms should be tested daily as hydrogen peroxide is unstable.
• Do not use blood agar as media containing whole red cells will contain catalase and could therefore give a
false positive result.
• The enzyme present in viable cultures only. Do not perform on cultures over 24 hours old. Older cultures may
give false-negative reactions as they are no longer viable
• A weak catalase (or pseudocatalase) reaction may be produced by some strains of aerococcus and
enterococcus species.

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• Hydrogen peroxide is unstable and should be stored in spark proof fridge. Avoid any undue exposure to light.
• Anaerobic cultures should be exposed to air for 30 minutes prior to testing.
• Use wooden applicator stick as inoculating loops or wires can react with the hydrogen peroxide to produce
false negative reactions.
7.9 References
1. Health Protection Agency BSOP TP 8
2. Monica Cheesbrough, Medical Laboratory Manual for Tropical Countries, Volume II, Microbiology. 1984.
Butterworth–Heinemam, Microbiology Edition, EL/BS with Tropical Health Technology, 1991, p 60–61

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7.10 Procedure Flowchart for Catalase Test

7.10.1 Method 1: Tube or Bottle Method
Place @ 0.2 mL of hydrogen

peroxide solution in a test tube
or Bijoux bottle.
Pick a colony to be tested with a loop along

with a positive and negative control.
Rub the colony to be tested onto the inside

wall of the tube or bottle above the surface of
hydrogen peroxide solution.
Cap the tube or bottle and lift it to allow hydro-

gen peroxide solution to cover the colony.
Is QC ok? No
Yes
Is there vigorous bubbling present?
Yes No
Organism is catalase Organism is catalase

positive negative
Report and document test results

and quality control results.

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7.10.2 Method 2: Agar Slant Method
Test overnight growth of an

organism on an agar slant along
with positive and negative controls.
Repeat test along with QC
Cap the tube or bottle and lift it to allow

hydrogen peroxide solution to cover the colony
Is QC ok? No
Yes
Yes No

positive negative

and quality control results

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7.10.3 Method 3: Cover-slip Method
Suspect colony is tested along with

a positive and negative control
Place one drop of hydrogen peroxide

solution on the slide
Take corrective action and

repeat test with controls
Spot a small amount of the colony onto the

center of a cover-slip
Invert a coverslip and place it on the drop of

hydrogen peroxide solution
Is QC ok? No
Yes
Yes No

positive negative


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7.11 Catalase Control Results

S. aureus Enterococcus
ATCC 25923 faecalis
ATCC 29212
Comments: ..................................................................................................................................

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8.0 Camp Test

8.1 Purpose
To ensure the correct validated procedure is followed for performing the Camp test. To produce accurate,
8.2 Introduction
The Camp test (acronym for Christie Alkins and Munch Paterson who reported this phenomenon in 1944) was
designed to aid in the identification of Streptococcus Group B (Streptococcus agalactiae). This test relies on the fact
that most Strep. agalactiae strains prduce a diffusible extracellular compound that will, in conjunction with a specific
Beta-hemolysin of Staphylococcus aureus, cause complete lysis of sheep red blood cells in an agar medium.

Materials Equipment
Test organism 35 –37ºC incubator
Relevant ATCC Control Organism
5% sheep blood agar plate
Staphylococcus aureus Beta-lysin producing strain
Bacteriology inoculating loop
8.5 Quality Control

Quality control organism should be tested with each batch of test organism to ensure that techniques and reagents
are acceptable.
Results are only validated if the expected results of the control organisms are observed.


Positive Control: Streptococcus agalactiae ATCC strain 13813
Negative Control: Enterococcus faecalis ATCC strain 29212
Known Staph aureus strain: ATCC strain 25923

• Streak a known Staphylococcus aureus strain across the centre of a 5% sheep blood agar plate.
• Streak the test organism at right angles to the Staphylococcus aureus inoculum line, avoiding touching the
inoculum.
• On a different area of the plate, inoculate Streptococcus agalactiae ATCC strain as a positive control and
Enterococcus faecalis ATCC strain is inoculated the same as the negative control as per the second step.
• Incubate at 35 –37ºC for 18–24 hours in any of the following: O2, CO2, or ANO2.

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• At the end of incubation, examine plates for arrow-head zone of haemolysis in the junction between
Staphylococcus aureus and test/control organism.

Positive result: arrow head zone of haemolysis at junction with Staphylococcus aureus.
Negative result: no arrow head zone of haemolysis present.
8.9 References
1. Monica Cheesbrough, Medical Laboratory Manual for Tropical Countries, Volume II Microbiology. 1984.
2. Mosby Howard B.J, Clinical and Pathogenic Microbiology, 2nd Edition, CB Mosby Company, 1994, St. Louis.

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8.10 Procedure Flow Chart for Camp Test
Inoculate a single line down

the centre of a 5% sheep blood agar
plate with Staphylococcus aureus
ATCC 25923
Take corrective action and

repeat test with controls
Inoculate test organism at right angles to
Staphylococcal inoculum. Avoid touching inoculum
Inoculate positive QC Streptococcus agalactiae

ATCC 13813 at right angles to the S. aureus
Inoculate negative QC Enterococcus faecalis

ATCC 29212 at right angles to the S. aureus inoculum
Incubate at 35–37ºC for 18–25 hours and

examine plates for presence off arrow head
zone of haemolysis
Is QC ok? No
Yes
Arrow head zone of haemolysis present?
Yes No
Positive Camp test Negative Camp test
Presumptive group B
Not group B Streptococcus
Streptococcus
Document QC and test results

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8.11 Camp Test Control Results

agalactiae faecalis
ATCC 13813 ATCC 29212
Comments: ..................................................................................................................................

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9.0 Coagulase Test

9.1 Purpose
To ensure the correct validated procedure is followed for performing the coagulase tube test, which
distinguishes between coagulase producing Staphylococcus aureus and other Staphylococcus species.
9.2 Introduction
Members of the genus Staphylococcus are differentiated by their ability to clot plasma by the action of the
enzyme coagulase. Coagulase binds plasma fibrinogen, causing the organism to agglutinate or clot plasma.
Coagulase exists in two forms: ‘bound coagulase’ (or clumping factor) which is bound to the cell wall and ‘free
coagulase’ which is liberated by the cell wall. The slide coagulase test detects bound coagulase while the tube
coagulase test detects both bound and free coagulase.
Bound coagulase absorbs fibrinogen from the plasma and alters it so it precipitates on the Staphylococci,
causing them to clump resulting in cell agglutination. Free coagulase reacts with a substance in plasma to form
a fibrin clot.

9.4 Media Reagents and Equipment
Reagents Required Materials/Equipment Required
Commercially available plasma with EDTA Test Organisms
Control Organisms ATCC strains.
Sterile clean glass tubes
Sterile wooden sticks
Timer
Disposable pipettes
Bacterial inoculating loop
Sterile water
Test tube rack
35–37ºC Incubator
Clean glass microscope slides
9.5 Quality Control

Control organisms should be tested with each batch of test organisms to ensure that the reagent is working
properly and that techniques and methods are acceptable.

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9.5.1 Quality Control Organism

Positive control: Staphylococcus aureus ATCC 25923
Negative control: Staphylococcus epidermidis ATCC 12228

9.6.1 Reagent Preparation
Rehydrate commercial coagulase plasma by adding sterile distilled water to the vial as indicated by
manufacturers instructions.
9.6.2 Tube Coagulase Procedure

1. Using a sterile 1ml pipette, add 0.5 ml of rehydrated coagulase plasma into a sterile test tube for each test
sample and quality control.
2. Using a sterile loop for each test sample and quality control, emulsify 2–4 discreet representative bacterial
colonies in the plasma.
3. Alternatively add 0.5 ml of the overnight broth culture of the organism to the tube of plasma.
4. Mix gently.
5. Incubate at 35–37ºC and examine hourly up to 4 hours.
6. Examine by gently tipping the tube.
7. Avoid shaking or agitating the tube, which will cause break down of the clot and consequently doubtful or
false negative test result.
8. Examine for a clot which gets the whole contents of the tube or forms a loose web of fibrin. If negative,
incubate overnight at 22–25ºC (room temperature) and re-examine at 24 hrs.
9.6.3 Slide Coagulase Procedure

1. Place a drop of distilled water on each end of a slide.
2. Emulsify a colony of the test organisms in each of the drop on the slide to make two thick suspensions.
3. Similarly emulsify a colony of each control in two drops of water on a slide.
4. Mix a loopful of plasma into one of the suspensions. No plasma is added to the second suspension. (This is
used to differentiate any autocoagulation from true coagulase clumping.)
5. Look for clumping of the organism within 10 seconds.

9.7.1 Slide Procedure
Positive: Visible clumping within 10 seconds
Negative: No visible clumping within 10 seconds
Negative and delayed results must be verified with the tube test.
9.7.2 Tube Procedure

Positive Results: Formation of a clot up to 4 hours at 37ºC or following overnight incubation.
Negative Results: No visible clot formation.

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9.8.1 Slide Coagulase Test
• Autoagglutination may occur.
• Use water instead of saline as some Staphylococci are salt sensitive, particularly if they have been cultured
in salt medium, and lysis or clumping of cells may occur.
• Over-mixing may cause the clot to break down.
9.8.2 Tube Coagulase Test

• Use EDTA as citrated plasma may be clotted by any organism that can utilize citrate.
• Incubating for longer than 4 hours at 37ºC may result in the disappearance of a clot. This is due to the production
of staphylokinase which can lyse the clot resulting in false negative results.
• Many weak enzyme producing strains will coagulate the plasma only after 24 hrs of incubation.
• Avoid shaking or agitating the tube, which will cause break down of the clot and consequently cause doubtful
or false negative test result
9.9 References
1. National Health Agency NHS BSOP TP 10i3
2. Howard BJ, Clinical and Pathogenic Biology, 2nd edition, CV Mosby Co. St Louis, 1994.

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9.10.a Procedure Flowchart for Slide Coagulase Test
Test organism along with

positive and negative QC
Place a drop of distilled water on the slide
With a loop, straight wire or wooden stick, emulsify the

test strain to obtain a homogenous thick suspension
Discontinue test
Observe for autoagglutination and perform the
tube coagulase
Is autoagglutination present? Yes
No
Mix a loopful of coagulase plasma into the suspension. Take corrective action
Observe for visible clumping within 10 seconds and repeat test
Is QC ok? No
Yes
Is visible clumping seen?
Yes No
Coagulase Positive Coagulase Negative

Staphylococcus species Staphylococcus species
Document and report test results along with QC

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9.10.b Procedure Flowchart For Tube CoagulaseTest
Test organism along with positive

and negative QC
Using a sterile 1ml pipette, add 0.5 mls of rehydrated

coagulase plasma to a sterile test tube

Using a sterile loop, emulsify 2–4 discreet bacterial
colonies in the tube of plasma. Mix gently
Incubate at 35 –37ºC and examine hourly up to 4 hours

and up to 24 hours at room temperature for any
degree of clot
Is QC ok? No
Yes
Is there clot formation at 4 hours or 24?
Yes No
Possible Other coagulase negative

Staphylococcus aureus Staphylococci
Document test results and quality

control results and report.

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9.11 Coagulase Control Results

Staphylococcus Staphylococcus
aureus epidermidis
ATCC 25923 ATCC12228
Comments: ..................................................................................................................................

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10.0 Clindamycin resistance in Staphylococcus species and Beta-hemolytic Streptococcus species

10.1 Purpose
To determine if Staphylococcus species and/or Beta hemolytic Streptococcus species are truly sensitive to
clindamycin and resistant to erythromycin
10.2 Introduction
When performing sensitivity testing using the Disk Diffusion method, some Staphylococcus and Beta hemolytic
Streptococcus may give a clindamycin sensitive and erythromycin resistant interpretative result. Normally
erythromycin should be sensitive and clindamycin should be resistant, unless there is enzymatic interference
affecting the actions of the drugs. In this case it is the Erm-gene that makes clindamycin appear to be sensitive
when it is actually resistant. In such cases the Clindamycin Resistance or ‘D’ Test is used to detect this
inducible clindamycin resistance.
This test applies to all staphylococcus and beta hemolytic streptococcus that exhibit clindamycin sensitive and
erythromycin resistant antibiotic susceptibility test results.

Reagents Supplies Equipment
Pure culture of a 16–24 hrs S. aureus Sterile Pasteur pipettes Discard pans/sharps
or Beta hemolytic streptococcus containers
Sterile Cotton swabs
grown on a suitable solid culture medi-
Nephelometer
um
(Vitex colorimeter)
Erythromycin 15μg disk
Bunsen Burner
Clindamycin 2μg disk
Vortex
Mueller Hinton broth
Mueller Hinton plates or tube media
0.85% sterile saline in tubes
QC strains
0.5 McFarland standard
10.5 Quality Control



Staphylococcus aureus ATCC 25923

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10.6 Examination
• Suspend organism in Mueller Hinton broth (MH) equal to 0.5 McFarland standard.
• Immerse a sterile non-toxic swab into standardized inoculum suspension.
• Rotate swab several times, pressing firmly on the inside wall of the tube above the fluid level to remove excess
inoculum.
• Streak swab over entire surface of Mueller Hinton agar plate for confluent growth.
• Repeat step 4 twice more, rotating the plate each time to ensure uniform growth.
• A final sweep is made on the agar rim with the swab.
• Inoculate the swab on a quadrant of the appropriate media to check for purity.
• Apply erythromycin and clindamycin disks to the agar approximately 15mm apart using sterile forceps measuring
with a ruler from the edge of one disk to the edge of the other. Gently press against disk to be sure it is in complete
contact with the agar.
• Incubate plate at 33–35ºC in an ambient air incubator for 24 hours.
• Examine plate closely using transmitted light for any indentation of growth (a ‘D’ shape).

10.7.1 Interpretation
• Blunting of the zone forming a ‘D’ shape around the clindamycin disk indicates resistance to clindamycin.
• Sometimes there may be a few colonies inside the clindamycin zone of inhibition on the clindamycin on the ‘D’
side, called ‘D+’. This is just an expression of another Erm gene causing induced clindamycin resistance.
• A light, hazy growth inside the entire zone of inhibition around the clindamycin dick with the ‘D’ effect is deemed
‘HD’ or ‘hazy D’; indicating another mechanism of clindamycin resistance.
10.7.2 Reporting
• If ‘D’ Test is positive (Inducible resistance to clindamycin detected). Also report erythromycin resistant and
clindamycin resistant in addition to the note below:
• This isolate is presumed to be resistant based on the detection of inducible clindamycin resistance.
Clindamycin may still be effective in some patients.
• If no inducible resistance to clindamycin detected. Report erythromycin resistant and clindamycin sensitive.
• If erythromycin is sensitive, inducible clindamycin resistance is not applicable and do not comment about
clindamycin induction on patient’s report.

• If both clindamycin and erythromycin are resistant by MIC/disk, then inducible clindamycin resistance is not
applicable.
• If either or both QCs fail, check reagents and disks for expiration dates. Also check the incubation temperatures,
purity of QC specimens and procedures for inconsistencies. Take corrective action and repeat test procedures.
• Never report results if the QCs fail.
10.9 References
1. Sutcliffe J., A. Tait-Kamradt, L. Wondrack. 1999. Streptococcus pneumoniae and Streptococcus pyogenes
resistant to macrolides but sensitive to clindamycin: a common resistance pattern mediated by an efflux
system. Antimicrob. Agents Chemother...40 (8): 1817–24.
2. Mechanisms of resistance to macrolides and lincosamides: nature of resistance elements and their clinical
implications. 2002. Clin. Infect. Dis. 34: 482–492.
3. NCCLS. Jan. 2004. Performance Standards for Antimicrobial Susceptibility Tests; Fourteenth Informational
Supplement. M100-S14. Vol. 24, No.1. Wayne, PA

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10.10 Flowchart For ‘D’ Test (Induced Clindamycin Resistance)
Staphylococcus or Beta
haemolytic streptococcus that is
Clindamycin sensitivite and
Erythromycin resistance
Make a 0.5 McFarland standard with pure freshly

cultured organisms and controls
Streak each test organism and controls from

0.5 McFarland suspensions onto a Mueller Hinton
agar plate for continuous growth Check disks expiration
dates, QC organisms,
procedure performed and
incubation temperature
On each Mueller Hinton plate put one Erythromycin
then repeat tests
and one Clindamycin disk 15mm apart using a
sterile forceps
Incubate plates at 33–35ºC in ambient

air incubator for 24 hours
Did controls pass? No
Yes
Is a ‘D’ shape around
Yes No
Report Clindamycin as resistant Report Clindamycin as Sensitive

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10.11 Bench (‘D’) (Induced Clindamycin Resistance) Control Results
Date Batch Positive Control Comments Sign

Staph aureus
NCTC 25923
Comments: ..................................................................................................................................

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11.0 Germ Tube Test

11.1 Purpose
The purpose of the Germ tube test is to detect the presence or absence of short filamentous extensions from
yeast cells. It is used to differentiate Candida albicans from all other species of Candida.
11.2 Introduction
Candida albicans is responsible for 80–90% of candidiosis. The remainders of infections are caused by other
species of Candida: Candida tropicalis; Candida krusei; Candida glabrata and Candida krusei. Candida species
are commensals in some sites of the body but proliferate under certain conditions, thereby causing infections.
Positive germ tube formation exhibits the short lateral filaments branching from the yeast cell. The only specie
of Candida to exhibit germ tube formations (short filamentous extensions which extend linearly from the yeast
cell) is Candida albicans. All other species of Candida will have a negative result.


• Bovine serum or human plasma
• Small test tubes
• Tube rack
• Pasteur pipettes
• Clean glass microscope slides
• Glass coverslips
• 37ºC Incubator
• Microscope with 40x objective



The Germ tube test is done using known commercially available controls of Candida albicans (ATCC 10231)
and Candida tropicalis (ATCC13803) or known in house controls if not available. Expected results of controls
validate patient results.
Expected Results:
Positive germ tube formation: Candida albicans.
Negative germ tube formation: Candida tropicalis.

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Positive and negative controls are set up and run alongside patient test.
1. Add 0.5ml (about 3 drops) bovine serum or human plasma to a clean small test tube.
2. Emulsify a colony of yeast in the serum or plasma using a sterile inoculating loop.
3. Place in tube rack and incubate for 2–4 hours at 37ºC.
4. Add a drop of the emulsification to a clean microscope slide and apply a glass coverslip on top.
5. Observe for germ tube formation under 40x objectives.

Short lateral filaments extending from the yeast cell are interpreted as germ tubes.
Positive: presence of germ tubes Candida albicans
Negative: absence of germ tubes other Candida species

Pseudohyphae formed by Candida tropicalis may be misinterpreted as germ tubes.
11.9 References
1. TML/MSH Microbiology Policy and Procedure Manual, Technical Manual, July 31,
2000, pg 56.
2. Murray PA , et al. Manual of Clinical Microbiology, 7th ed, 1999, pp1189–1191.

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11.10 Procedure Flowchart For The Germ Tube Test
Add 0.5 ml human plasma a small

test tube.Prepare same for positive
and negative control
Emulsify a colony of yeast in plasma

And repeat test with
controls
Incubate at 37ºC for 2– 4 hours
Apply a drop of the emulsification onto a clean,

microscope slide and place a coverslip on top
Is the Quality Control ok? No
Yes
Are germ tubes observed?
Yes No
Candida albicans Other species of Candida
Report and document results of

both patient and quality controls

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11.11 Germ Tube Control Results

Candida albicans Candida tropicalis
ATCC 10231 ATCC 13803
Comments: ..................................................................................................................................

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12.0 India Ink Test

12.1 Purpose
The India Ink Test is performed to provide rapid results to aid in the diagnosis of infections caused by the yeast,
Cryptococcus neoformans. It is used to provide reliable, accurate and reproducible results having clinical utility.
12.2 Introduction
Meningitis infections caused by Cryptococcus neoformans are opportunistic infections usually seen in
immunocompromised patients. It is sporadic yeast which may usually affect the lungs, brain and meninges and
other body sites, may also be associated with respiratory infections. It is associated with HIV infection and
diseases of the reticuloendothelial system. A cerebrospinal fluid sample is obtained by a special procedure
called a lumbar puncture. This specimen is treated as urgent and is tested immediately upon receipt.
The Cryptococcus neoformans yeast is surrounded by a large gelatinous polysachharide capsule. This thick
capsule makes the yeast cell hard to identify from stained smears. India ink therefore is a test which enables the
technician to observe the encapsulated yeast (about 2–15μm in diameter) are observed against a dark background.
Light reflects from the yeast allowing one to observe the clear halo around the yeast cell or budding cells.

Please note that Cryptococcus neoformans is a category 3 pathogen, therefore if suspected all isolates
must be handled in a Biosafety Cabinet.
Safety must be in accordance with safety guidelines for handling of specimens and reagents as outlined in the
laboratory safety manual.
All work likely to generate aerosols must be done under a microbiological safety cabinet.
12.4 Reagents and Equipment
Reagents Materials Equipment
India Ink Reagent Clean glass microscope slides Microscope w/ 40x and 100x
objectives
Glass coverslips
Inoculatine wire/loop

Quality Control should be run concurrently with tests.
Quality control is performed to ensure validity, accurate and reproducible results and to ensure expected
performance of the reagents used for the test.


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Negative control: Candida albicans ATCC 10231

12.6.1 From Colony
Each patient test should be done along with negative controls.
1. Place a small drop of India ink on a clean glass microscope slide.
2. Using a sterile wire/loop, emulsify a touch of the colony to be tested in the 50% aqueous India ink. Mix
gently.
3. Apply a coverslip on to create a thin preparation
4. Examine under 40 x objectives for Cryptococcal yeast cells observed with a clear halo, against a dark
background.
12.6.2 From Cerebrospinal Fluid

12.7.1 From Colony
Negative result: Candida albicans ATCC 10231
Validate results along with quality controls. If quality controls are invalid, corrective action must be taken and
test must be repeated along with quality controls. Immediately report and document both patient results and
quality controls.
12.7.2 From Cerebrospinal Fluid

Negative result: Cryptococcus species or other specie of yeast
Validate results along with quality controls. If quality controls are invalid, corrective action must be taken and
test must be repeated along with quality controls. Immediately report and document both patient results and
quality controls.
12.8 Precautions/Limitations of Procedure

The polysaccharide capsule surrounding the yeast will not be easily seen if the preparation is too thick.
12.9 References
1. Monica Cheesbrough, Medical Laboratory Manual for Tropical Countries, Volume II Microbiology. 1984. pg
390–391.

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12.10 Procedure Flowchart for India Ink Test
Add a small drop of 50% aqueous

India ink reagent onto clean
microscope glass
Aseptically emulsify a touch of the colony to be

tested or a loopful of CSF sediment. Mix
Repeat test along with
controls
Place a glass coverslip on top for a thin preparation

Examine under 40X for yeast cells surrounded by
a clear halo
Is Quality Control ok? No
Yes
Are yeast cells observed with clear halo?
Yes No
Other Cryptococcus
Cryptococcus neoformans
species or other yeast
Document test results and

QC results and report

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12.11 India Ink Stain Control Results
Date Batch Negative Control Comments Sign

Candida albicans
ATCC 10231
Comments: ..................................................................................................................................

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13.0 Indole Test

13.1 Purpose
To ensure the correct validated procedure is followed for performing the Indole test producing accurate,
13.2 Introduction
The indole test detects tryptophanase production and aids in the differentiation of enterobactericeae and other
genera.
The indole test determines the ability of the organism to produce indole from the degradation of the amino acid
tryptophan. Tryptophan is hydrolyzed by tryptophinase to produce three possible end products, one of which
is indole. A coloured product is produced when indole is combined with certain aldehydes, e.g. Kovac's reagent.
Two methods are described, a spot method which detects rapid indole producing organisms and the conventional
tube method requiring overnight incubation which identifies weak indole producing organisms. For the tube
method, tryptophan (peptone broth) can be used or one of the biochemical test medium namely SIM or MIL.


Safety must be in accordance with safety guidelines for handling of specimens and reagents as outlined in the
laboratory safety manual. All work likely to generate aerosols must be performed in microbiological safety
cabinet.
• Hydrochloric acid is corrosive.
• Kovac’s indole reagent is an irritant.

13.4.1 Tube Method
• 1% Tryptophan or peptone broth or suitable biochemical Kovac’s reagent
• Kovac’s reagent (for use with broth/semisolid media cultures)
• p-dimethylaminobenzaldehyde 10.0g
• Amyl alcohol150ml
• Concentrated HCL 50ml
Dissolve benzaldehyde in amyl alcohol (may require warming to 50ºC) then carefully add the acid. If reagent turns
red, place in refrigerator at 4ºC until colour changes to golden yellow. Store at 4ºC in a brown bottle covered with
aluminum foil (to prevent breakdown of reagent by light).
13.4.2 Spot Method (for use with plate cultures)

• p-dimethylamino benzaldehyde 1.0g
• M/10 HCL100ml
• Dissolve aldehyde in the acid and store as recorded for tube method.
• Reagent for spot method can also be commercially obtained.
• Bacteriological straight wire/loop (preferably nichrome) or disposable alternative

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Positive control: Escherichia coli ATCC 25922 red colour
Negative control: Pseudomonas aeruginosa ATCC 27853 yellow colour

13.6.1 Tube Method (broth or semisolid medium)
• Inoculate broth/medium with test organisms and incubate at 37ºC for 18–24 hrs.
• Add .5ml/3 drops Kovac’s reagent and gently agitate.
• Examine the upper layer of liquid/medium.
Positive result: red colour
Negative result: yellow colour
13.6.2 Spot Method

• Moisten a piece of filter paper with spot test reagent.
• Smear a colony of test organism on surface.
• Examine immediately.
Positive result: Green/blue colour change
Negative result: No colour change

13.7.1 Tube Method (broth or semisolid medium)
• Inoculate broth/medium with test organisms and incubate at 37ºC for 18–24 hrs.
• Add .5ml/3 drops Kovac’s reagent and gently agitate.
• Examine the upper layer of liquid/medium.
Positive result: red colour
Negative result: yellow colour
13.7.2 Spot Method

• Moisten a piece of filter paper with spot test reagent.
• Smear a colony of test organism on surface.
• Examine immediately.
Positive result: Green/blue colour change

Negative result: No colour change

1. If peptone is used instead of tryptophan broth, the batch should be checked with a positive control to ensure
adequate indole production from the peptone.
2. Organisms to be tested by the spot method must be taken from a tryptophan containing medium (e.g.
blood agar)

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3. Do not use peptone medium with added glucose as acid production may inhibit indole production.
4. Cultures to be tested for indole must be incubated aerobically (as far as possible). A decrease in oxygen
tension decreases indole production.
13.9 References
1. Murray, PR, et at; Manual of Clinical Microbiology, 7thEdition, 1999, pp1668.

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13.10.1 Procedure flowchart for Indole Test Tube Method
Inoculate tryptophan/peptone
broth or media e.g. MIL, SIM
Incubate overnight at
37 ºC aerobically
Add Kovac’s reagent
Take corrective
What is the result?
action
Red / bright pink

Yellow colour
colour
Positive Negative
Is QC ok?
Yes No
Record and report result

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13.10.2 Procedure Flowchart for Indole Test Spot Method
Moisten filter paper with

spot test reagent
Smear test organism on paper
Examine immediately for result/colour
Take corrective
What is the result?
action
Blue/green colour No colour change
Positive Negative
Is QC ok?
Yes No
Record and report result

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13.11 Indole Control Results

E. coli Pseudomonas
ATCC 25922 aeruginosa
ATCC 27853
Comments: ..................................................................................................................................

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14.0 Novobiocin Susceptibility Test

14.1 Purpose
To ensure the correct validated procedure is followed for performing the Novobiocin susceptibility test, to
produce accurate reliable reproducible results having clinical utility.
14.2 Introduction
This test is used to differentiate Staphylococcus saprophyticus from coagulase negative Staphylococci
isolated from urine specimens. Of the clinically significant coagulase negative staphylococci; Staphylococcus
saprophyticus is resistant to 5 μg of Novobiocin. A diameter of inhibition of 16mm or less around a 5μg
Novobiocin is one of the clinically significant CNS and this disc constitutes a resistant test result and gives a
presumptive identification of Staphylococcus saprophyticus.

Materials Equipment
Mueller Hinton agar plates Incubator
Bacteriological inoculating needles Bacti-incinerator
Quality Control organisms
Test organism
5μg Novobiocin disc
0.5 Mc Farland standard
Sterile cotton swabs
Sterile tryptic soy broth

• Examine Mueller Hinton plates for signs of contamination before use.

• Examine plates with control organism to ensure a pure growth of organism.
• Control organism should be tested with each batch of test organisms to ensure the validity of test results.
• Results are only valid if expected results of the control organism are observed.
• Expired or discoloured discs and/ or plates must not be used.

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14.5.1 Quality control organism:

Positive control: Staphylococcus saprophyticus ATCC 49453
Neagative control : Staphylococcus epidermidis ATCC 12228

1. Prepare a 0.5 Mc Farland standard of test and control organisms in sterile 0.85% saline or sterile Tryptic Soy
Broth. Commercially prepared 0.5 Mc Farland standards are available.
2. Use a sterile bacteriological needle to inoculate test organisms in saline or tryptic soy broth to 0.5 McFarland
turbidity standard.
3. Dip a sterile cotton swab into the suspension. Within 15 minutes the swab should be rotated several times
and pressed firmly on the inside wall of the tube above the fluid level.
4. Inoculate the entire surface of a Mueller Hinton plate using the swab
5. After the plate is dry place a 5 μg Novobiocin disc in the center of the inoculum using sterile forceps
6. Incubate the plate at 35–37ºC for 18–24 hours.
7. Measure the diameter of the zone of inhibition using a ruler following incubation.

Resistant Sensitive: Staphylococcus saprophyticus (zone of inhibition of <16 mm)
Other coagulase negative staphylococci (zone of 17 mm or greater)

N/A
14.9 References
1. Monica Cheesbrough, Medical Laboratory Manual for Tropical Countries, Volume II Microbiology. 1984.
2. Mosby Howard B.J, Clinical and Pathogenic Microbiology, 2nd Edition, CB Mosby Company, 1994, St. Louis.

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14.10 Procedure Flowchart For Novobiocin Susceptibility Test
Prepare 0.5 Mc Farland Standard

of the test and control organism in
0.85 % saline or tryptic soy broth
Inoculate test organism in saline or tryptic soy broth

to a 0.5 McFarland turbidity standard
Within 15 minutes of adjusting the turbidity, dip

sterile cotton swab into the suspension
Rotate swab several times and pressed firmly on the

inside wall of the tube above the fluid level
Use the swab to inoculate the entire surface of a

Mueller Hinton plate
After the plate is dry, use sterile forceps to place a 5ug

Novobiocin disc in the center of the inoculum
Incubate the plate at 35– 37ºC for 18–24 hours
Following incubation, measure the diameter

of the zone of inhibition using a ruler
No Take corrective
Is QC ok? action and repeat
test with controls
Yes
Is there a zone of inhibition

measuring 16 mm or less?
Yes No
Staphylococcus Other Coagulase negative

saprophyticus Staphylococcus species
Document test and quality

control results and report

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14.11 Novobiocin Sensitivity Control Results

Staphylococcus Staphylococcus
saprophyticus epidermidis
ATCC 49453 ATCC12228
Comments: ..................................................................................................................................

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15.0 Optochin Test

15.1 Purpose
To ensure the correct and validated procedure is followed for processing of the optochin test, to provide
accurate, reliable, reproducible results having clinical validity.
15.2 Introduction
Susceptibility to optochin (ethylhydrocupriene hydrochloride) is a simple and reliable method of differentiating
Streptococcus pneumoniae from other alpha-hemolytic streptococci.
The optochin test detects an organisms susceptibility to the chemical optochin (ethylhydrocupriene
hydrochloride). The chemical tests the fragility of the bacterial cell membrane and causes S. pneumoniae to
lyse due to changes in surface tension.
15.3 Safety Instruction


The optochin test is widely used in the form of filter paper discs, impregnated with ethylhydrocupriene
hydrochloride which are applied directly to inoculated plates before incubation.
Filter paper discs impregnated with 5μg of ethylhydrocupriene hydrochloride.

Bacteriological straight wire/loop (preferable nichrome) or disposable alternative.


15.5.1 Quality Control organisms

Positive control: Streptococcus pneumoniae ATCC 19615
15.6 Examination Procedures

15.6.1 Pure colony
• Streak a blood plate with the organism to be tested along with the positive and negative controls.
• Place an optochin disc in the centre of the inoculum.
• Incubate at 35–37ºC for 18–24 h in 5% CO2
• Examine for zones of inhibition.
15.6.2 Specimen
• Streak the specimen onto a blood plate.
• Place an optochin disc in the centre of the primary inoculum.
• Incubate at 35–37ºC for 18–24 h in 5% CO2
• Examine for zones of inhibition.

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Positive result: zone of inhibition of ≥5 mm radius from the edge of the disc. i.e. test organism is Streptococcus
pneumoniae
Negative result: no zone of inhibition, or a zone <5 mm radius from the edge of the disc i.e. test organism is
not Streptococcus pneumoniae.

• Some viridans streptococci may produce a small zone of inhibition. Equivocal results should be confirmed
using the bile solubility test or by the use of commercial kit.
• False negative results may be reported if cultures are in high concentrations of CO2
• Occasional strains of optochin resistant Streptococcus pneumoniae have been reported.
15.9 References
1. Health Protection Agency BSOP TP 25.

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15.10 Procedure Flowchart For Optochin Test
Test organism along with

positive and negative control
Streak blood agar plates with organism to be tested
Place optochin disc in the centre of the inoculum
Take corrective
Is QC ok?
No action
Yes
Are there zones of inhibition?
Yes No
If zones of inhibition of If zones of inhibition of

≥5 mm radius from the ≤5 mm radius from the
edge of the disc edge of the disc
A negative result for

A positive result for
optochin indicates the
optochin identifies
organism is not
S. pneumoniae
S. pneumoniae
Document QC and test results

and report

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15.11 Optochin and Penicillin Sensitivity Control Results

pneumoniae faecalis
ATCC 19615 ATCC 29212
Comments: ..................................................................................................................................

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16.0 Oxidase test

16.1 Purpose
To ensure the correct validated procedure is followed for performing the oxidase test producing accurate,
reliable, reproducible results having clinical utility
16.2 Introduction
The test is used to differentiate pseudomonads from other non-fermentative species of bacteria. The test is
used to aid in the differentiation of Neisseria, Moraxella, Campylobacter and Pasteurella species, all being
oxidase positive. The oxidase test is used to determine if an organism possesses the cytochrome oxidase
enzymes.
Oxidase positive bacteria possess cytochrome oxidase or independent oxidase (an iron containing
haemoprotein). These both catalyse the transport of electrons from donor compounds (NADH) to electron
acceptors (usually oxygen).
The test reagent N,N,N,’N’-tetra-methyl-p-phenylenediamine dihydrochloride acts as an artificial electron

acceptor for the enzyme oxidase. The oxidase reagent forms the coloured compound indophenol blue.
The cytochrome system is usually only present in aerobic organisms which are capable of utilizing oxygen as
the final hydrogen receptor. The end product of this metabolism is either water or hydrogen peroxide (broken
down by catalase).


• 24 hr (fresh) growth of colonies on non-selective solid medium
• 1% oxidase reagent in distilled water or commercial dropper reagent/impregnated oxidase test strips.
• Bacteriological straight wire/loop or disposable alternative
• Filter paper
• Pasteur pipettes/droppers
• Commercial preparations are available
• Quality Control organisms


16.5.1 Quality control organisms

Positive control: Pseudomonas aeruginosa ATCC 27853
Negatve control: Escherichia coli ATCC 25922

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16.6.1 Filter Paper Method 1
• Soak a piece of filter paper with reagent solution
• Scrape some fresh growth from plate with sterile/ disposable loop or stick and rub onto the filter paper
• Examine for blue colour within 10 seconds
16.6.2 Filter Paper Method 2

• Soak a piece of filter paper in reagent solution and allow paper to dry.
• Touch a colony with a piece of the paper
16.6.3 Direct Plate Method (do not use on colonies intended for sub-culture)
• Add 1–2 drops of reagent to suspect colonies
16.6.4 Swab Method (do not use on colonies intended for sub-culture)
• Add 1–2 drops reagent to swab or dip in reagent
• Touch colony with swab
16.6.5 Impregnated oxidase strip method

• Scrape some fresh growth from plate with sterile/disposable or stick
• Rub onto filter test strip

Positive result: development of blue colour
Negative result: no blue colour

• The test should not be performed on cultures from media containing tellurite and fermentable carbohydrates
as these may prevent the reaction occurring.
• Results from old cultures are unreliable.
• Do not use nichrome inoculating loops or wires. False positive reaction may occur due to surface oxidation
products formed during flame sterilization.
• Some Pseudomonas species are oxidase-negative.
• The test solution auto-oxidizes rapidly – use a fresh solution or add 1% ascorbic acid to retard oxidation. Do
not use if the solution is blue. Control test every solution.
16.9 References
1. Murray, PR et al; Manual of Clinical Microbiology 7th ed., 1999 pp 1670

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16.10 Procedure Flowchart for Oxidase Test
Perform at the same time

as the test
Dip swab into

Soak filter paper with reagent
reagent
Add 1– 2 drops of
reagent to suspected
colonies on plate
Touch suspected
colony on plate
Add colonies to filter
paper or commercial
impregnated test strip
Is there a colour change?

Investigate, take
corrective actions
and repeat
Blue colour No colour change
Positive Negative
Did QC pass?
Yes No

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16.11 Oxidase Control Results

Pseudomonas E. coli
aeruginosa ATCC 25922
ATCC 27853
Comments: ..................................................................................................................................

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17.0 PYR Test

17.1 Purpose
To ensure the correct validated procedure is followed for performing the PYR test producing accurate, reliable,
reproducible results having clinically utility.
17.2 Introduction
PYR (L-pyrrolidonyl-B-naphthylamide) serves as a substrate for the detection of pyrrolidonyl peptidase. Following
the hydrolysis of the substrate by the enzyme, the resulting B-naphthylamine produces a red colour upon the
addition of P-dimethylaminocinnamaldehyde reagent (PYR regent). This test is used in conjunction with others, for
the identification of catalase negative, gram positive cocci including Group A Streptococci and Enterrococci.

Reagents Materials
PYR disks(L_pyrrodidonyl-_-naphthylamide) Glass slides
PYR reagent 0.01% P-dimethylaminocinnamaldehyde Sterile Petri dish
Bacteriological inoculating loop
Forceps
Sterile distilled water
17.5. Quality Control


Positive Control – Group A Streptococcus: Streptococcus pyogenes ATCC 19615
Negative Control – Group B streptococcus: Streptococcus agalactiae ATCC 13813

1. Place a PYR disk/s onto a glass slide/Petri dish and moisten it with a drop of sterile distilled water.
2. Rub a loopful of the culture onto disk, holding it in place with sterile forceps
3. Leave at room temperature for 2 minutes
4. After 2 minutes, add 1 drop PYR reagent

Result:
Positive: Pink or cherry red colour within one minute
Negative: No colour change or slight yellow colour

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17.8 Limitations and Pitfalls of the Procedure

N/A
17.9 References
1. Murray, PR, et at; Manual of Clinical Microbiology, 7th Edition, 1999, pp286.
2. Remel kit insert information.

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17.10 Procedure Flowchart for PYR Test
Place PYR disc onto glass slide

or petri dish
Moisten with sterile distilled water
Rub a loopful of culture and disc, holding disc in Investigate, take

place with sterile forceps corrective actions
and repeat
Leave at room temperature for 2 minutes
Is there a colour change?
Pink or cherry Slight yellow/

red No
Positive Negative
Does the QC pass?
Yes No

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17.11 PYR Control Results

pyogenes agalactiae
ATCC 19615 ATCC 13813
Comments: ..................................................................................................................................

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18.0 Urease test

18.1 Purpose
To ensure the correct, validated procedure is followed for processing the urease test to provide accurate, reliable,
reproducible results having clinical utility
18.2 Introduction
The urease test is use to differentiate urease-positive Proteus species from other members of the
Enterobacteriaceae. Some strains of Enterobacter and Klebsiella species are also urease-positive. This test
also differentiates the urease-negative Corynebacterium diphtheriae from the urease-positive Corynebacterium
ulcerans and Corynebacterium pseudotuberculosis. Helicobacter pylori and most Brucella species are urease-
positive. Bacteroides ureolyticus split urea rapidly, usually within a few minutes, and is a quick way of
identifying this organism. The urease test may aid in the identification of Cryptococcus species which produces
a positive result after prolonged incubation.
The urease test is used to determine the ability of an organism to split urea by production of the enzyme urease.
Two units of ammonia are formed producing alkalinity. The production of alkali is detected by a pH indicator.
Christensen’s urea contains the pH indicator phenol red which under acid conditions (pH 6.8) is yellow. In alkaline
conditions (pH 8.4) the indicator turns the media rose pink.


• Discrete colonies growing on solid medium.
• Christensen’s medium or liquid alternative. A commercially available alternative may be used according to the
manufacturer’s instructions.
• Bacteriological straight wire/loop (preferably nichrome) or disposable alternative.


Positive control: Proteus mirabilis ATCC 7002
Negative control: Escherichia coli ATCC 25922

Quality control should be carried out on each batch of media before it is used, and every time the test is performed.
• Use QC organisms to ensure that the media is working properly
• Label the positive and negative urease agar tube
• The two tubes are inoculated with QC organism as indicated. Inoculate organism heavily over the entire
surface. The tubes are placed in racks in an air incubator at 35–37ºC for 18–24h and checked for growth and
colour change after this time
• Incubate inoculated slope at 35–37ºC in a water bath or hot block or incubator
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• Examine slopes after 4h and after overnight incubation
• If the medium turns purple/pink colour, it is a positive result. If the colour of the medium remains unchanged, that
is a negative result.
• The tubes are placed in racks in an air incubator at 35–37ºC for 18–24h and observed for growth and colour
change after this time
Positive result: purple/pink colour

Negative result: colour of medium remains unchanged
18.8 Limitations & Pitfalls
18.9 References
1. Health protection Agency BSOP TP 36

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18.10 Procedure Flowchart For Urease Test

and negative controls
Inoculate slope heavily over the entire surface
Incubate inoculated slope at 35 –37ºC for 18–24 hr. Take corrective

action
Examine slopes up to 4 hours and after

overnight incubation
Is QC ok? No
Yes
Is there a colour change to bright pink?
No No
Urease Test is Urease Test is

Positive Negative
Document and report test results


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18.11 Urease Control Results

Proteus mirabilis E. coli
ATCC 7002 ATCC 25922
Comments: ..................................................................................................................................

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19.0 X and V test

19.1 Purpose
To ensure the correct, validated procedure is followed for differentiation of Haemophilus species by the X and V
test, to provide accurate, reliable, reproducible results having clinical utility.
19.2 Introduction
This SOP describes the differentiation of the Haemophilus species by the X and V test.
Species of the genus Haemophilus require either or both of the two factors X and V for growth. X factor is
compromised of protoporphyrin IX, haemin or other iron-containing porphyrins. These are required for growth
because X-dependent strains are unable to convert α-amino-laevulinic acid to protoporphyrin. V factor
compromises nicotinamide adenine dinucleodide phosphate (NADP). Both factors are present in blood.
The factors are incorporated into filter paper discs which are placed on a blood-free medium previously
inoculated with the organism under test.


Discrete bacterial colonies growing on solid medium
19.4.1 Test agar

Oxoid blood agar base CM 55
Commercial X, V and XV discs
Bacteriological straight wire/loop (preferably nichrome) and disposable alternative
19.5 Quality control


19.5.1 Quality control organisms

X and V factor: Haemophilus influenzae ATCC 35056
V factor: Haemophilus parainfluenzae ATCC 33392

• Make a light suspension of the test organism by touching one or more colonies with a straight wire and
emulsifying in normal saline
• Soak a swab in the suspension and spread evenly across the entire surface of a nutrient agar plate
• Position X, V and XV discs on the agar surface. Ensure the discs are a minimum of 3.5 cm apart in an equilateral
triangle configuration to prevent diffusion from the discs giving false results.
• Incubate in 5% CO2 at 35–37º C overnight.

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Examine the plates in a good light source for growth around the discs. Growth indicates a requirement for that
particular factor
The presence or absence of growth around the discs is recorded.
Interpretation of Results
X factor V Factor XV factor
H. influenzae No Growth No Growth Growth
H. haemolyticus No Growth No Growth Growth
H. parainfluenzae No Growth Growth Growth
H. aphrophilus Growth Growth Growth
H. paraphrophilus No Growth Growth Growth
H. segnis No Growth Growth Growth

• The V factor diffuses more readily than the X factor. If the discs are placed too close together, the V factor
may diffuse towards the X factor disc, leading to growth apparently due to X factor rather than V.
• Care must be taken to avoid carryover of blood from the medium when ‘picking’ colonies. This will also lead
to erroneous results.
• Commercial manufacturers of X and V discs do not specify the concentration of the factors. Acceptance of
a batch of discs must be based on an ‘in use’ performance test with a range of Haemophilus species rather
than an essay of content.
• No nutrient agar is entirely deficient in X factor and the disc test may be erroneous in up to 20% of cases,
usually ascribing H. influenzae to H. parainfluenzae.
• More accurate results are obtained with the porphyrin synthesis test (BSOP TP 29)
19.9 References
1. Health Protection Agency: BSOP TP 38

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19.10 Procedure Flowchart For X and V Factor Test

and negative control
Make a light suspension of the test organism
Soak a sterile swab in the suspension Spread evenly across

the surface of a nutrient agar plate
Aseptically place X, V, and XV Factor Discs on the surface of

the agar, about 3-5 cm apart in an equilateral triangle
Take corrective
action and repeat
test with controls
Incubate in 5% CO2 at 35–37ºC overnight.
Examine for growth around discs
No
Are QC ok?
Yes
Is there any growth?
Growth only around

the XV factor disc Growth only around V
and XV factor discs
Result may be
H. influenzae Result is presumptive
H. parainfuenzae
Proceed to further biochemical

testing for H.influenzae type B
Document test and QC results

and report.

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19.11 X and V Factor Control Results
Date Batch Positive Control Comments Sign

Haemophilus influenzae
ATCC 9006
Comments: ..................................................................................................................................

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20.0 Ziehl–Neelsen Stain Test

For more comprehensive instructions, refer to CRM-SOP 1: Microscopy of sputum to detect AAFB
Please note that Mycobacterium species are Category 3 pathogens and should be handled as such.
20.1 Purpose
To ensure the correct validated procedure is followed for performing the Zeihl–Neelsen test to produce accurate,
20.2 Introduction
The ZN Stain is used to stain smears prepared primarily from sputum specimens from patients suspected of having
tuberculosis. It is commonly used as a screening tool for early detection and to initiate treatment in a presumptive
diagnosis of mycobacterial infection e.g. Pulmonary tuberculosis.
The main characteristics of mycobacterium are that they are acid fast, because of the high concentration of
lipo-polysaccharide in their cell wall. They hold the aniline dye, basic fuschin, and are difficult to decolorize. They
retain the red colour even after treatment with a mixture of acid-alcohol. Methylene blue or Malachite green maybe
used as the counter stain in the technique.

Preparation and staining of AFB slides should be performed in a Biosafety Cabinet. All specimens for AFB
investigation must be handled with caution. All PPE must be used at all times.

• Carbol Fuschin
• 3% Acid–Alcohol
• Methylene blue/Malachite green
• Slides
• Staining rack
• Swabs/Inoculating loop
• Bunsen burner/ Slide warmer
• Microscope with oil immersion lens
• (Stains may be obtained in kits or prepared in-house).
Specimen Preparation
• Apply specimen to a clean, dry labeled slide using swab or loop. Be careful not to make smear too thick
• Sputum, spinal fluid or other body fluid are suitable specimens for a smear
• Allow smear to air dry
• Fix smear by passing through low flame 2–3 times, or leave on slide warmer. Be careful not to overheat slide


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Control slides can be prepared in-house by using known positive patient samples and the negative control using a
suspension of Escherichia coli equivalent to #1 McFarland standard. However, QC slides are commercially available
and should be purchased in preference to preparing in-house slides due to the Health & Safety risk involved in their
preparation.
• Positive & negative controls should be stained with each batch of test slides.
• The quality of stains and procedure must be monitored.
• A new batch of stains should be checked with controls before use.

Mycobacterium tuberculosis ATCC 25177 – AFB positive
Bacillus subtilis ATCC 6633 – AFB negative
or Escherchia coli ATCC 25922 – AFB negative

1. Place slide on staining rack and flood with carbol fuschin
2. Gently heat slide to steaming, remove Bunsen burner and allow steaming for 5 minutes.
3. Wash gently in running water
4. Decolorize with acid/ alcohol for 1–2 minutes or until no more red colour appears in washing
5. Wash gently in running water.
6. Counter stain with methylene blue/ malachite green
7. Wash gently in running water
8. Allow to air dry
9. Examine using oil immersion lens
20.7 Interpretationa and Reporting

Reporting AFB positive – Red bacilli seen
AFB negative: No red bacilli seen
20.7.1 Suggested Quantitative Reporting

more than 9 AFB/ field – 4+
1–9 AFB/ field – 3+
1–9 AFB/ 10 fields – 2+
1–9 AFB/ 100 fields – 1+
1–2 AFB/ 300 fields – Doubtful repeat
No AFB seen – Negative

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• For in vitro diagnostic use only.
• The reagents used are harmful and total if swallowed and can cause eye irritation.
• The reagents can cause irritation also to the skin and respiratory system.
• Protective gears especially gloves should be used while doing staining smear.
• Hands should be thoroughly washed after using stain.
• Avoid insufficient decolorizing with acid–alcohol.
• Staining times should be adhered do precisely. Not to do so can lead to over or under decolourisation/staining
therefore giving false positive or negative results.
• Most mycobacterium are strongly acid fast and are not easily decolorized.
• Smears that are too thick are difficult to decolorize and when counter stained may acid mask the presence of
fast bacilli.
• The counter stain should not be so intense that it ‘hides’ the mycobacterium.
• Beware of false-positive smears. Common sources of AFB which can contribute to this problem are:
1. Tap water and infrequently cleaned distilled water reservoirs. Check these sources from time to time to see if
AFB can be detected.
2. Transfer of material from slides in bulk staining tanks. To avoid this stain slides individually.
3. Transfer of ‘positive’ flakes from thick smears to other smears via oil immersion lens between slides; allow oil
to free fall from applicator onto smear instead of touching the applicator to the slide.
4. Mycobacterium culture should be followed by smear evaluation of specimen to confirm diagnosis and give
AST results for treatment.
20.9 Reference
1. Kit package insert
2. Ridley M.J., Ridley D.S. Stain techniques and morphology of mycobacterium lepta–leprosy
3. Jokahaski S. Handbook of direct smear examination of sputum for tubercle bacillus, SE AMIC publication, No.
4, 1975.
4. I Collins CH, Grange JM, Yates MD. Tuberculosis Bacteriology 2nd Edition, 1997
5. Isenberg, Henry D, Essential Procedures for Clinical Microbiology ASM Press 1998,
pp 176–178
6. Murray, PR et al; Manual of Clinical Microbiology, 7th ed., 1999 pp 1678.

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20.10 Procedure Flowchart for Ziehl–Neelson (ZN) Stain
Prepare test smears along with

control slides and heat
Flood slides with Carbol Fuschin stain and steam

for 5 minutes
Check reagents,
expiration dates,
Wash gently with cold running water procedures and
prepare fresh slides.
Allow to dry and
repeat procedure
Decolorize with acid-alcohol for 1–2 minutes until
no more red appears
Wash gently with cold running water

No
Stain with counter stain (Malachite green or

Methylene blue) for 30 seconds to 1 minute
Did both positive and negative

Wash gently with cold running water
controls pass?
Air dry and examine microscopically
Yes
Yes
Are red bacilli seen?
No
(Quantitative) Acid Fast Bacilli seen No Acid Fast Bacilli seen

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20.11 ZN Stain Control Results

Mycobacterium Bacillus subtillis
tuberculosis ATCC 6633
ATCC 25177 or E. coli ATCC 25922
Comments: ..................................................................................................................................

88

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CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURES

Quality Control of Reagents and Tests – SOP No: CRM-SOP 21

CAREC PAHO WHO EUROPEAN UNION

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN

1.0 Reagent and Test Quality Control 6

2.0 Process Flow Chart For Quality Control of Bacteriology Tests 10

3.0 Record Chart for Quality Control of Reagents and Stains 11

4.0 Bacitracin Differentiation Test 12

5.0 Beta-Lactamase (Cefinase) Test 16

6.0 Bile Solubility Test 20

7.0 Catalase Test 25

8.0 Camp Test 32

9.0 Coagulase Test 36

10.0 Clindamycin resistance in Staphylococcus species and

11.0 Germ Tube Test 44

12.0 India Ink Test 50

13.0 Indole Test 54

14.0 Novobiocin Susceptibility Test 60

15.0 Optochin Test 64

16.0 Oxidase test 68

17.0 PYR Test 72

18.0 Urease test 76

19.0 X and V test 80

20.0 Ziehl–Neelsen Stain Test 84

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN

Country Name Organisation

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN

Country Name Organisation

Function Name Organisation

Graphic Design and Support

Function Name Country

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN

Controlled Document Reference CRM-SOP 21

Controlled Document Title Standard Operating Procedure for Quality Control of

Amendment Issue Number Insert Issue Page Section(s) Amendment

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN

1.0 Reagent and Test Quality Control

1.1 Test and Reagent Quality Control Procedures

1.5 Staff Competency Requirements

1.6 Safety Instructions

Samples and culture plates should be autoclaved before finally discarding.

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN

1.7 Pre-Examination Procedure

1.7.2 Sample Collection

1.7.3 Transport & Storage

1.7.4 Rejection criteria

1.7.5 Relevant clinical information

1.8 Media, Reagents and Equipment

1.9 Examination Procedures

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN

1.9.1 Quality Control

Refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

Refer to departmental SOPs for further details.

Refer to CRM-SOP 7: Gram Stain for methodology

Refer to CRM-SOP 22: Inoculation of Culture Media

Refer to CRM-SOP 7: Gram Stain for methodology

1.9.5 Susceptibility testing

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN

1.10 Post-Examination Procedures

1.10.2 Sample Retention, Storage and Disposal

1.11 Limitations and Pitfalls

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN

2.0 Process Flow Chart For Quality Control of Bacteriology Tests