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close this bookBasic Laboratory Procedures in Clinical Bacteriology (WHO; 1991; 128 pages)
View the documentPreface
View the documentIntroduction
close this folderQuality assurance in microbiology
View the documentIntroduction
View the documentDefinitions
View the documentInternal quality control
View the documentExternal quality assessment
open this folder and view contentsPart I. Bacteriological investigations
open this folder and view contentsPart II. Essential media and reagents for isolation and identification of clinical pathogens
View the documentSelected further reading
View the documentSelected WHO publications of related interest
View the documentBack Cover
 

Internal quality control

Requirements

An internal quality control programme should be:

 

• practical
• realistic
• economical

An internal quality control programme should not attempt to evaluate every procedure, reagent, and culture medium on every working day. It should evaluate each procedure, reagent, and culture medium according to a practical schedule, based on the importance of each item to the quality of the test as a whole.

Procedures

Internal quality control begins with proper laboratory operation.

Laboratory operations manual

Each laboratory should have an operations manual, covering the following subjects:

 

• cleaning of the working space,
• personal hygiene,
• separation of working areas from eating and smoking areas,
• safety precautions,
• handling and disposal of infected material,
• appropriate immunization,
• care of equipment,
• collection of specimens,
• registration of specimens,
• elimination of unsuitable specimens,]
• processing of specimens,
• recording of results,
• reporting of results.

The operations manual should be carefully followed, and regularly revised and updated.

Care of equipment

It is particularly important to take good care of laboratory equipment. Good quality tests cannot be performed if the equipment used is either of poor quality or poorly maintained.

Table 1 is a schedule for the routine care and maintenance of essential equipment. Equipment operating temperatures may be recorded on a form such as the one shown in Fig. 2.

Table 1. Quality control of equipment

Equipment

Routine care

Monitoring

Technical maintenance and inspection

Anaerobic jar

Clean inside of jar each week
Reactivate catalyst after each run (160 °C, 2h)
Replace catalyst every 3 months

Use methylene blue indicator strip with each run
Note and record decolorization time of indicator each week

Inspect gasket sealing in the lid weekly

Autoclave

Clean and change water monthly

Check and adjust water level before each run
Record time and temperature or pressure for each run
Record performance with spore-strips once a week

Every 6 months

Centrifuge

Wipe inner walls with antiseptic solution weekly or after breakage of glass tubes or spillage

 

Replace brushes annually

Hot-air oven for sterilization of glassware

Clean inside monthly

Record time and temperature for each run

Every 6 months

Incubator

Clean inside walls and shelves monthly

Record temperature at the start of each working day (allowance 35 ± 2°C)

Every 6 months

Microscope

Wipe lenses with tissue or lens paper after each day’s work
Clean and lubricate mechanical stage weekly
Protect with dust cover when not in use

Check alignment of condenser monthly

Annually

Refrigerator

Clean and defrost every 2 months and after power failure

Record temperature on first day of each week (allowance 2-8 °C)

Every 6 months

Water-bath

Wipe inside walls and change water monthly

Check water level daily
Record temperature on first day of each week (allowance 54-57 °C)

Every 6 months

Culture media

Culture media may be prepared in the laboratory from the basic ingredients, or from dehydrated powders available commercially, or may be purchased ready for use. Commercial dehydrated powders are recommended because they are economical to transport and store, and their quality is likely to be higher than media prepared in the laboratory. For best results, careful attention is required to the points itemized below.

Selection of media

An efficient laboratory stocks the smallest possible range of media consistent with the types of test performed. For example, a good agar base can be used as an all-purpose medium, for preparing blood agar, chocolate agar, and several selective media.


Fig. 2. Record of equipment operating temperature

One highly selective medium (Salmonella/Shigella agar or deoxycholate citrate agar) and one less selective medium (MacConkey agar) are generally all that is necessary for the isolation of pathogenic Enterobacteriaceae from stools.

A special culture medium should be added for the recovery of Campylobacter jejuni.

Ordering and storage of dehydrated media

1. Order quantities that will be used up in 6 months, or at most 1 year.

2. The overall quantity should be packed in containers that will be used up in 1 - 2 months.

3. On receipt, tighten caps of all containers securely. Dehydrated media absorb water from the atmosphere. In a humid climate, seal the tops of containers of dehydrated media with paraffin wax (fill the space between the lid and container with molten wax, and let it harden).

4. Write date of receipt on each container.

5. Store in a dark, cool, well ventilated place.

6. Rotate the stock so that the older materials are used first.

7. When a container is opened, write the date of opening on it.

8. Discard all dehydrated media that are either caked or darkened.

9. Keep written records of media in stock.

Preparation of media

1. Follow strictly the manufacturer’s instructions for preparation.

2. Prepare a quantity that will be used up before the shelf-life expires (see below).

Storage of prepared media

1. Protect against sunlight.

2. Protect against heat. Media containing blood, other organic additives, or antibiotics should be stored in the refrigerator.

3. The shelf-life of prepared media, when stored in a cool, dark place, will depend on the type of container used. Typical shelf-lives are:

 

- tubes with cotton-wool plugs, 3 weeks;
- tubes with loose caps, 2 weeks;
- containers with screw-caps, 3 months;
- Petri dishes, if sealed in plastic bags, 4 weeks.

Quality control of prepared media

1. pH testing. The pH of the prepared medium need not be checked routinely when it is correctly prepared from dehydrated powder. If the medium is prepared from basic ingredients, it should be allowed to cool before the pH is tested. Solid media should be tested with a surface electrode or after maceration in distilled water. If the pH differs by more than 0.2 units from the specification, adjust with acid or alkali or prepare a new batch.

2. Sterility testing. Carry out routine sterility tests on media to which blood or other components have been added after autoclaving. Take 3 - 5% of each batch and incubate at 35 °C for 2 days. Refrigerate the rest. If more than two colonies per plate are seen, discard the whole batch.

3. Performance testing. The laboratory should keep a set of stock strains for monitoring the performance of media. A suggested list of stock strains is given in Table 2. These strains can be obtained through routine work, or from commercial or official sources.

Table 2. Suggested stock strains for quality controla

Gram-positive cocci

Enterobacteriaceae

Enterococcus faecalis (ATCC 29212 or 33186)

Citrobacter freundii

 

Enterobacter cloacae

Staphylococcus aureus (ATCC 25923)

Escherichia coli (ATCC 25922)

Staphylococcus epidermidis

Klebsiella pneumoniae

Streptococcus agalactiae

Proteus mirabilis

Streptococcus mitis

Salmonella typhimurium

Streptococcus pneumoniae

Serratia marcescens

Streptococcus pyogenes

Shigella flexneri

Gram-negative fastidious organisms

Yersinia enterocolitica

Branhamella catarrhalis

Other Gram-negative rods

Haemophilus influenzae type b

Acinetobacter calcoaceticus biovar Iwoffi

 

β-lactamase negative

Pseudomonas aeruginosa (ATCC 27853)

 

β -lactamase positive

Vibrio cholerae (non-01)

Haemophilus parainfluenzae

Fungi

Neisseria gonorrhoeae

Candida albicans

Neisseria meningitidis

 

Anaerobes

 

Bacteroides fragilis

 

Clostridium perfringens

 

 

a The strains most relevant to the needs of the laboratory should be selected.

A list of performance tests for commonly used media is given in Table 3.

The procedures to be followed when carrying out performance tests on new batches of media are:

 

• Prepare a suspension of the stock strain with a barely visible turbidity, equivalent to that of the barium sulfate standard used in the modified Kirby-Bauer method (McFarland 0.5) and use 1 loopful as inoculum.

• Incubate for the length of time used routinely. Read the plates in the usual way.

• Keep proper records of results.

Stains and reagents

Recommendations for testing a number of reagents are given in Table 4. Testing should be carried out:

 

• each time a new batch of working solution is prepared;

• every week (this is critical for the cold Ziehl-Neelsen stain: the classical stain has a shelf-life of several months).

Stains and reagents should be discarded when:

• the manufacturer’s expiry date is reached;

• visible signs of deterioration appear (turbidity, precipitate, discoloration).


Diagnostic antigens and antisera

In order to obtain the best results from antigens and antisera:

 

• Always follow the manufacturer’s instructions.

• Store at the recommended temperature. Some serological reagents do not tolerate freezing.

• Avoid repeated freezing and thawing. Before freezing, divide antiserum into aliquots sufficient for a few tests.

• Discard when the manufacturer’s expiry date is reached.

• To test agglutinating antisera, always use pure, fresh cultures of known reactivity.

• Always include a serum control of known reactivity in each batch of tests. The serum may be from a patient, or from a commercial source.

• If possible, the potency of the control serum should be expressed in International Units per ml.

• Paired sera from the same patient, taken during the acute and convalescent phases of the disease, should be tested with the same batch of reagents.

• For the serological diagnosis of syphilis, only nationally or internationally recognized procedures should be used.

• Each batch of serological tests should include:

 

- a negative serum (specificity control);
- a weakly reactive serum (sensitivity control);
- a strongly reactive serum (titration control), which should read within one dilution of its titre when last tested.

 

• Always record all control serum titres.

Table 3. Performance tests on commonly used media

Medium

Incubation

Control organism

Expected result

Bile-aesculin agar

24 h

E. faecalis

growth and blackening

   

S. pyogenes

no growth

Blood agar

24 h, CO2 (candle jar)

S. pyogenes

growth and β-haemolysis

   

S. pneumoniae

growth and α-haemolysis

Chocolate agar

24 h, CO2

H. influenzae

growth

Decarboxylase (cover with sterile oil)

     

- lysine

48 h

S. typhimurium

positive

   

S. flexneri

negative

- ornithine

48 h

S. typhimurium

positive

   

K. pneumoniae

negative

- arginine (dihydrolase)

48 h

S. typhimurium

positive

   

P. mirabilis

negative

Gelatinase (rapid tests)

24 h

E. coli

negative

   

S. marcescens

positive

Kligler iron agar (see triple sugar iron agar)

     

MacConkey agar with crystal violet

24 h

E. coli

red colonies

   

P. mirabilis

colourless colonies (no swarming)

   

E. faecalis

no growth

Malonate broth

24 h

E. coli

negative (green)

   

K. pneumoniae

positive (blue)

Mannitol salt agar

24 h

S. aureus

yellow colonies

   

S. epidermidis

rose colonies

   

E. coli

no growth

Methyl red/Voges-Proskauer

48 h

E. coli

positive/negative

   

K. pneumoniae

negative/positive

Mueller-Hinton agar

24 h

E. coli ATCC 25922

acceptable zone sizes (see Table 13)

   

S. aureus ATCC 25923

 
   

P. aeruginosa ATCC 27853

 

Nitrate broth

24 h

E. coli

positive

   

A. calcoaceticus

negative

Oxidation/fermentation (OF) dextrose (without oil)

24 h

P. aeruginosa

oxidation at the surface

   

A. calcoaceticus biovar Iwoffi

no change

Peptone water (indole)

24 h

E. coli

positive

   

K. pneumoniae

negative

Phenylalanine deaminase

24 h

E. coli

negative

   

P. mirabilis

positive

Salmonella-Shigella agar or deoxycholate citrate agar

24 h

E. coli

no growth

   

S. typhimurium

colourless colonies

   

Y. enterocolitica

colourless colonies

   

S. flexneri

colourless colonies

Selenite broth

24 h

S. typhimurium

growth after subculture

   

E. coli

no growth after sub culture

Simmons citrate (incubate with loose screwcap)

48 h

E. coli

no growth

   

K. pneumoniae

growth, blue colour

Thiosulfate citrate bile salts (TCBS) agar

24 h

Vibrio spp (non-agglutinable)

yellow colonies

Tetrathionate broth (as for selenite broth)

 

E. coli

no growth

Thayer-Martin agar

24 h, CO2

N. meningitidis

growth

   

N. gonorrhoeae

growth

   

Staphylococcus spp

no growth

   

E. coli

no growth

   

C. albicans

no growth

Thioglycollate broth

24 h

B. fragilis

growth

Triple sugar iron agar (depth of butt should be at least 2.5 cm; incubate with loose screw-cap)

24 h

C. freundii

A/A gasa+H2S

   

S. typhimurium

A/A gasa+H2S

   

S. flexneri

K/Aa

   

A. calcoaceticus

no change

Urea medium

24 h

E. coli

negative

   

P. mirabilis

positive (pink)

Voges-Proskauer (see methyl red/Voges-Proskauer)

     

 

a A/A: acid slant; K/A: alkaline slant.

Table 4. Performance tests on commonly used reagents

Reagent or stain

Species suitable for testing

Medium

 

Positive

Negative

 

Bacitracin disc

S. pyogenes (zone)

E. faecalis

blood agar

Catalase

S. aureus

E. faecalis

TSAa

Coagulase plasma

S. aureus

S. epidermidis

TSA

β-glucuronidase (PGUA)

E. coli

K. pneumoniae

TSA

Gram stain

Staphylococcus

E. coli

mixed in smear

ONPGb

S. marcescens

S. typhimurium

triple sugar iron agar or Kligler agar

optochin discs

S. pneumoniae (zone)

S. mitis

blood agar

oxidase

P. aeruginosa

E. coli

TSA

tellurite disc

E. faecalis (no zone)

S. agalactiae (zone)

blood agar

V-factor (discs or strips)

H. parainfluenzae

H. influenzae

TSA

XV-factor (discs or strips)

H. influenzae

 

TSA

Ziehl-Neelsen stain

M. tuberculosis

mixed non-acid-fast flora

sputum smearc

 

a Tryptic soy agar.

bO-Nitrophenyl-β-D-galactopyranoside.

c Prepare a number of smears from known positive and negative patients. Fix by heat, wrap individually in paper, store in the refrigerator.

Antibiotic susceptibility tests

The routine use of the modified Kirby-Bauer method is recommended. To avoid errors, the following guidelines should be used:

 

• Discs should be of correct diameter (6.35 mm).

• Discs should be of correct potency (see Table 13).

• The stock supply should be stored frozen (- 20°C).

• The working supply should be kept no longer than I month in a refrigerator (2-8 °C).

• Only Mueller-Hinton agar of performance-tested quality should be used.

• Correct pH (7.2 - 7.4) of the finished medium is essential for some antibiotics.

• The inoculum should be standardized against the prescribed turbidity standard.

• Zone sizes should be measured exactly.

• Zone sizes should be interpreted by referring to a table of critical diameters. Zone diameters for each test organism should fall within the limits given in Table 14.

• Only pure cultures of rapidly growing bacteria give reliable readings.

• The three standard control strains1 are:

 

- Staphylococcus aureus (ATCC 25923; NCTC 6571);
- Escherichia coli (ATCC 25922; NCTC 10418);
- Pseudomonas aeruginosa (ATCC 27853; NCTC 10622).

1 These strains can be obtained from: (ATCC) American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, USA; or (NCTC) National Collection of Type Cultures, Public Health Laboratory Service, London NW9, England.

 

• Tests should be carried out with the three standard strains:

 

- when a new batch of discs is put into use;
- when a new batch of medium is put into use;
- once a week, in parallel with the routine antibiograms.

 

• Use the quality control chart shown in Fig. 11 for recording and evaluating performance tests.

Maintenance and use of stock cultures

Selection and origin

Select the strains so that the maximum number of morphological, metabolic, and serological characteristics can be tested with the minimum number of cultures; a suggested list is given in Table 2. These strains can be obtained from a combination of the following sources:

 

- properly documented isolates from clinical specimens;
- official culture collections;
- commercial producers;
- external quality assessment surveys;
- reference laboratories.

Preservation

Long-term preservation

Long-term preservation methods permit intervals of months or even years between subcultures. The best methods are lyophilization (freeze-drying), or storage at - 70 °C or below, in an electric freezer or in liquid nitrogen. Alternative methods are described below.

1. Glycerol at -20°C.

 

• Grow a pure culture on an appropriate solid medium.
• When the culture is fully developed, scrape it off with a loop.
• Suspend small clumps of the culture in sterile neutral glycerol.
• Distribute in quantities of 1-2 ml in screw-capped tubes or vials.
• Store at -20°C.
• Avoid repeated freezing and thawing.
• Transfer after 12-18 months.

2. Mineral oil at room temperature.1

 

• Prepare tubes of heart infusion agar with a short slant. For fastidious organisms, add fresh native or heated blood. Sterilize mineral oil (liquid petrolatum) in hot air (170°C for 1 hour). Grow a pure culture on the agar slant.

• When good growth is seen, add sterile mineral oil to about 1 cm above the tip of the slant.

• Subculture when needed by scraping growth from under the oil.

• Store at room temperature.

• Transfer after 6-12 months.

1 MORTON, H. E. & PULASKI, E. J. The preservation of bacterial cultures. Journal of bacteriology, 38: 163 -183 (1938).

3. Stab cultures at room temperature (use for non-fastidious organisms only, such as staphylococci and Enterobacteriaceae).

 

• Prepare tubes with a deep butt of carbohydrate-free agar. Tryptic soy agar (soybean casein digest agar) is recommended.

• Stab the organism into the agar.

• Incubate overnight at 35 °C.

• Close tube with screw-cap or cork.

• Dip cap or cork into molten paraffin wax to seal.

• Store at room temperature.

• Transfer after 1 year.

4. Stab cultures in cystine trypticase agar (CTA) (for Neisseria and streptococci).

 

• Prepare tubes of CTA basal medium.
• Stab the organism into the medium.
• Incubate overnight at 35 °C.
• Close tube with screw-cap or cork.
• Dip cap or cork into molten paraffin to seal.
• For Neisseria, store at 35 °C, and transfer every 2 weeks. For streptococci, store at room temperature, and transfer every month.

5. Cooked-meat medium for anaerobes.

 

• Inoculate tubes.
• Incubate overnight at 35 °C.
• Close tube with screw-cap or cork.
• Store at room temperature.
• Transfer every 2 months.

Short-term preservation

Working cultures for daily routine tests can be prepared in the following ways.

1. Rapid-growing organisms

 

• Inoculate on tryptic soy agar slants in screw-capped tubes.
• Incubate overnight at 35 °C.
• Store in refrigerator.
• Transfer every 2 weeks.

2. Streptococci

 

• Inoculate on blood agar slants in screw-capped tubes.
• Incubate overnight at 35 °C.
• Store in refrigerator.
• Transfer every 2 weeks.

3. Meningococci and Haemophilus

 

• Inoculate on chocolate agar slants or plates.
• Incubate overnight at 35 °C.
• Store at room temperature.
• Transfer twice a week.

4. Gonococci

 

• Inoculate on chocolate agar.
• Incubate and store at 35 °C.
• Transfer every 2 days.
• Replace the quality control strain by each new clinical isolate.

Use of reference laboratories

The following categories of specimen should be submitted to a regional or central reference laboratory:

 

• Specimens for infrequently requested or highly specialized tests (e.g., virology, serodiagnosis of parasitic infections).

• Occasional duplicate specimens, as a check on the submitting laboratory’s own results.

• Specimens needing further confirmation, specification, grouping, or typing of pathogens of great public health importance, e.g., Salmonella, Shigella, Vibrio cholerae, Brucella, meningococci, and pneumococci.

Reference laboratories should be able to supply reference cultures for quality control and training needs, and standard sera and reagents for comparison with those in use in the referring laboratory.

If no external quality assessment programme exists, the reference laboratory should be asked to supply blind, coded specimens and cultures so that the referring laboratory may test its own proficiency in isolation and identification.

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