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close this bookHealth and Environment in Sustainable Development - Five Years after the Earth Summit (WHO, 1991, 128 p.)
close this folderPart II. Essential media and reagents for isolation and identification of clinical pathogens
close this folderPriority grading of pathogens, culture media, and diagnostic reagents
View the document(introduction...)
View the documentBlood culture
View the documentCerebrospinal fluid specimens
View the documentUrine
View the documentStool culture
View the documentLower respiratory tract
View the documentUpper respiratory tract
View the documentUrogenital specimens for agents of sexually transmitted diseases
View the documentPus and exudates
View the documentList of recommended culture media and diagnostic reagents for the intermediate microbiological laboratory

(introduction...)

A certain degree of flexibility has been incorporated by adopting a graded priority code for pathogens, media, and diagnostic reagents, as follows:

Code 1: high priority
Code 2: intermediate priority
Code 3: low priority.

Pathogen priority

The categorization of pathogens is based on a number of factors: frequency of isolation, clinical relevance, severity of disease, epidemic potential, and the cost-benefit ratio of isolation and/or identification. As an example of the latter factor, chlamydiae, although considered as important pathogens, receive low priority, since there is no cheap laboratory technique for their detection. The grading is by no means absolute and will vary from country to country, or from laboratory to laboratory, depending on the local disease pattern, the laboratory capacity, and the resources available.

Media and reagents priority

The grading of media and reagents is linked to that of the pathogens for which they are used for isolation or identification. However, there may be differences - if the medium or reagent is used very broadly, it may score higher than any one pathogen for which it is used.

Code 1: High priority media and reagents should be available in all the laboratories that practise general diagnostic bacteriology. These are most often general-purpose, easy to prepare, and few in number.

Code 2: Intermediate priority media and reagents are additional useful substances that make laboratory diagnosis more complete and more useful for epidemiological studies, although they may not be essential for immediate patient care, e.g., the grouping antisera for meningococci.

Code 3: Low priority media and reagents are substances that are of value only occasionally for the patient, but which are useful in teaching, research, and specialized reference laboratories. This category applies to media and reagents that are too expensive for general use, or which are needed for organisms that rarely occur or are difficult to isolate and are therefore probably of low cost-effectiveness.

The pathogens involved, and the culture media and diagnostic reagents required, are listed below for some common bacteriological investigations, together with a suggested priority coding. The coding should be adapted for each laboratory according to local circumstances.

Blood culture

Common pathogens

Pathogens with high priority

Salmonella typhi and non-typhi
other Gram-negative rods
Neisseria meningitidis streptococci (S. pyogenes, S. pneumoniae, viridans streptococci)
Staphylococcus aureus

Pathogens with intermediate priority

Haemophilus influenzae
Bacteroides fragilis
Pseudomonas aeruginosa

Pathogens with low priority

Candida albicans and Cryptococcus neoformans
Brucella
Pseudomonas pseudomallei
non-fermenters other than Pseudomonas aeruginosa

Culture media and diagnostic reagents

Blood-culture broth

Priority code

tryptic soy broth (TSB) (can be replaced by any rich broth - for example, brain heart infusion broth)

1

optional: addition of sodium polyanethol sulfonate (SPS), 0.25 g per litre “anaerobic” blood-culture broth:

2


thioglycollate broth or Schaedler broth or



Wilkins-Chalgren anaerobe broth


Isolation media


subculture on blood agar, chocolate agar, and MacConkey agar

1

Identification media and reagents


oxidase reagent

1

optochin disc, bacitracin disc

1

b-lactamase test reagent

1

V and XV factors

2

Neisseria meningitidis agglutinating serum (polyvalent and specific groups A, B, C)

3

Haemophilus influenzae type b antiserum

3

coagulase plasma

1

Salmonella agglutinating antisera

1

Cerebrospinal fluid specimens

Common pathogens

Pathogens with high priority

Neisseria meningitidis
Streptococcus pneumoniae
Haemophilus influenzae
Gram-negative rods
Mycobacterium tuberculosis
Cryptococcus neoformans

Pathogens with intermediate priority

Streptococcus agalactiae (group B)

Pathogens with low priority

Listeria monocytogenes

Culture media and diagnostic reagents

Isolation media

Priority code

blood agar (with streak of Staphylococcus)

1

chocolate agar

1

MacConkey agar

1

Sabouraud dextrose agar

2

Lstein-Jensen medium

2

Identification reagents


oxidase reagent

1

optochin discs

1

b-lactamase test reagent

1

V and XV factors

2

Neisseria meningitidis agglutinating serum (polyvalent and specific groups A, B, C)

3

Haemophilus influenzae type b antiserum

3

Rapid diagnostic tests


test kit for rapid diagnosis of meningitis agents

3

Urine

Common pathogens

Pathogens with high priority

Escherichia coli
other Gram-negative rods
enterococci
Staphylococcus saprophyticus

Pathogens with intermediate priority

Pseudomonas and other non-fermenters
other staphylococci

Pathogens with low priority

Candida albicans
Mycobacterium tuberculosis

Isolation media and diagnostic reagents

Isolation and quantitative media

Priority code

brolacin agar (can be replaced by purple lactose agar, MacConkey agar without crystal violet, or eosin methylene blue agar)

1

Identification media and reagents


bile-aesculin agar (for enterococci)

2

novobiocin (5-mg) disc for differentiating coagulase-negative staphylococci

3

b-glucuronidase tablet (PGUA) for identification of E. coli

2

for Gram-negative rods:

1


Kligler iron agar

1


motility-indole-urease medium (MIU)

1


Kovac’s reagent for indole

1


oxidase reagent

1


ONPG test

2


Simmons’ citrate agar

2


cystine decarboxylase broth (Mr)

2

for staphylococci and enterococci:



coagulase plasma

1


catalase test (H2O2)

1


oxidase reagent

1


hydrogen generator for anaerobic jar

2

Stool culture

Common pathogens

Pathogens with high priority

Salmonella typhi and S. paratyphi
Shigella
Vibrio cholerae serogroup 01, noncholerae vibrios, Aeromonas and Plesiomonas

Pathogens with intermediate priority

non-typhoid salmonellae and Edwardsiella
Yersinia enterocolitica
Campylobacter jejuni
Clostridium difficile

Pathogens with low priority

Escherichia coli (enteropathogenic, enterotoxigenic, enteroinvasive, and enterohaemorrhagic)

Culture media and diagnostic reagents

Transport media

Priority code

Cary-Blair medium (for all pathogens)

1

buffered glycerol saline (not for Vibrio and Campylobacter)

2

Enrichment media


selenite F broth

2

alkaline peptone water

2

Isolation media


MacConkey agar (with crystal violet)

1

deoxycholate citrate agar (can be replaced by Salmonella-Shigella agar)

1

TCBS agar (can be replaced by Monsur’s TTG agar)

1

Campylobacter medium: Columbia agar base or any blood agar base with lysed blood and antibiotic supplement

2

Clostridium difficile selective medium

3

Preliminary identification media and reagents


Kligler iron agar (KIA) (may be replaced by triple sugar iron agar (TSI) but only for enteric pathogens)

1

motility-indole-urease medium (MIU) (may be replaced by motility test medium + peptone urea broth)

1

Kovac’s reagent for indole

1

oxidase reagent

1

Specific identification media and reagents


Biochemical


ONPG test (disc)

2

lysine decarboxylase broth (Mr base)

2

Simmons’ citrate agar

2

Andrade peptone water (or phenol red broth base)

2

acetate agar

3

DNase agar

3

vibriostatic compound 0/129 disc

3

Serological: agglutinating antisera


Priority code

Salmonella:

polyvalent 0

1

S. typhi:

anti OD (9, 12), anti H: d, anti Vi

1

S. typhimurium:

anti 0: 4, 5 (B), anti H: i

2

Shigella:

polyvalent flexneri, sonnei, boydii, dysenteriae, specific anti-Shigella dysenteriae 1 (Shiga)

1

Vibrio:

polyvalent 0:1

1


specific anti-ogawa and anti-inaba

3

Lower respiratory tract

Common pathogens

Pathogens with high priority

Mycobacterium tuberculosis
Streptococcus pneumoniae
Haemophilus influenzae
Staphylococcus aureus
Klebsiella pneumoniae

Pathogens with intermediate priority

Enterobacteriaceae
Candida albicans
Branhamella catarrhalis

Culture media and diagnostic reagents

Isolation media


blood agar

1

chocolate agar

1

MacConkey agar

1

Lstein-Jensen medium

2

Sabouraud dextrose agar

3

selective blood agar for Haemophilus (bacitracin or vancomycin)

3

Identification reagents


optochin disc

1

V and XV factors

2

coagulase plasma

1

Upper respiratory tract

Common pathogens

Pathogens with high priority

Streptococcus pyogenes (group A) (throat)
Corynebacterium diphtheriae (throat and nose)
Streptococcus pneumoniae (ear and sinus)
Haemophilus influenzae (ear and sinus)

Pathogens with intermediate priority

Candida albicans (oropharynx)
Staphylococcus aureus (ear and sinus)
Branhamella catarrhalis (ear and sinus)

Pathogens with low priority

Gram-negative rods and Pseudomonas (ear)
Neisseria meningitidis (nasopharyngeal carriers)

Culture media and diagnostic reagents

Isolation media

Priority code

blood agar (prepared from a carbohydrate-free base)

1

chocolate agar

2

Ler coagulated serum or Dorset egg medium

2

tellurite blood agar

2

modified Thayer-Martin medium (for gonococci and meningococci)

3

Identification reagents


bacitracin disc, optochin disc

1

catalase and coagulase reagents

1

carbohydrate degradation media for Neisseria

2

V and XV factors (discs or strips)

2

Rapid diagnostic kits


grouping kit for haemolytic streptococci

3

Urogenital specimens for agents of sexually transmitted diseases

Common pathogens

Pathogens with high priority

Neisseria gonorrhoeae
Treponema pallidum (dark-field microscopy)

Pathogens with intermediate priority

Haemophilus ducreyi
Gardnerella vaginalis (microscopic examination)
Candida albicans (microscopic examination)
Trichomonas vaginalis (microscopic examination)

Pathogens with low priority

Chlamydia trachomatis

Culture media and diagnostic reagents

Transport media

Priority code

Amies transport medium or Stuart transport medium

1

Isolation media


modified Thayer-Martin (MTM) medium or New York City (NYC) medium

1

Mueller-Hinton chocolated horse blood agar + vancomycin + IsoVitaleX for H. Ducreyi

3

Diagnostic reagents


oxidase reagent

1

Nitrocefin test or other b-lactamase test reagent

1

Pus and exudates

Common pathogens

Pathogens with high priority

Streptococcus pyogenes
Staphylococcus aureus

Pathogens with intermediate priority

Enterobacteriaceae
Pseudomonas and other non-fermenters
Clostridium perfringens
Bacteroides and other strict anaerobes
Streptococcus (other species)

Pathogens with low priority

Bacillus anthracis
Mycobacterium tuberculosis, M. ulcerans, etc.
Pasteurella multocida

Culture media and diagnostic reagents

Isolation media

Priority code

blood agar

1

MacConkey agar

1

mannitol salt agar

2

thioglycollate broth (with indicator) (can be replaced by cooked meat medium)

2

tryptic soy broth (TSB)

1

Identification media and reagents


coagulase plasma

1

catalase test (H2O2)

1

oxidase reagent

1

hydrogen generator for anaerobic jar

2

List of recommended culture media and diagnostic reagents for the intermediate microbiological laboratory

Culture media

Recommended medium

Alternative

Priority code

acetate agar


3

Andrade peptone water

phenol red broth base

2

bile-aesculin agar


1

blood agar base (see tryptic soy agar)

Columbia agar base

1

brolacin agar

purple lactose agar

1

decarboxylase broth base (Mr)


2

DNase agar


3

Kligler iron agar (KIA)


1

Ler coagulated serum

Dorset egg medium

1

Lstein-Jensen medium base


1

MacConkey agar (with crystal violet)


1

mannitol salt agar (MSA)


2

motility-indole-urease medium (MIU)

motility test medium + urea broth + peptone (tryptone) water

1

Mueller-Hinton agar


1

Sabouraud dextrose agar


1

deoxycholate citrate agar

Salmonella-Shigella agar (SS)

1

selenite F broth


2

Simmons’ citrate agar


2

tryptic soy agar (TSA)


1

tryptic soy broth (TSB)

brain heart infusion broth

1

thioglycollate medium (with indicator)

Schaedler broth, Wilkins-Chalgren anaerobe broth, cooked meat medium

2

TCBS

Monsur’s TTG medium

1

transport medium (Amies)

transport medium (Stuart or Cary-Blair)

1

Inhibitors or antibiotics for use in media or as reagents

Priority code

bacitracin (for isolation of Haemophilus spp)

3

Campylobacter antibiotic supplement

2

chloramphenicol (for isolation of fungi)

1

tellurite solution (for isolation of Corynebacterium diphtheriae)

2

vancomycin (for isolation of Haemophilus ducreyi or H. influenzae)

3

gonococcal antibiotic supplement: vancomycin, colistin, nystatin (trimethoprim): VCN (VCNT)

1

Clostridium difficile antibiotic supplement for selective agar

3

Enrichments for culture media


IsoVitaleX (Polyvitex, Vitox, supplement B, supplement VX, supplement CVA)

2

sodium-polyanethol sulfonate (SPS)

3

Diagnostic discs, tablets, or strips


b-glucuronidase (PGUA)

2

bacitracin

1

novobiocin (5 mg)

3

ONPG

1

optochin

1

oxidase

1

V, XV growth factors

2

vibriostatic compound 0/129

3

Nitrocefin disc (Cefinase) or reagent

1

Diagnostic kits


Rapid serodiagnostic kit for identification of meningococci

3

Serological grouping kit for haemolytic streptococci

3

Miscellaneous diagnostic reagents


barium sulfate standard (for Kirby-Bauer method)

1

buffered glycerol-saline (for transportation of stool)

2

Gram stain reagents

1

hydrogen peroxide (for catalase)

1

India ink (for capsule detection)

2

Kovac’s reagent (for indole)

1

oxidase reagent (dimethyl-p-phenylenediamine)

1

plasma (for coagulase test and germ tube test)

1

Ziehl-Neelsen stain

1

carbohydrates: glucose, lactose, maltose, mannitol, sucrose

2

amino acids: lysine (for decarboxylase test)

2

hydrogen generator for anaerobic jar

2

Sensitivity-testing discs

Antibiotics included in the WHO list of essential drugs (1990)

ampicillin
chloramphenicol
co-trimoxazole (sulfamethoxazole - trimethoprim)
erythromycin
gentamicin
nitrofurantoin
oxacillin
benzylpenicillin
piperacillin
sulfonamide
tetracycline (or doxycycline)
trimethoprim

Reserved antibiotics

amikacin
ceftriaxone
cefuroxime
cefalotin
ciprofloxacin or other fluoquinolones
clindamycin
nalidixic acid
tobramycin

Diagnostic antisera


Priority code

Salmonella:

polyvalent o

1


specific O-group: 9, 12 (D) and Vi

1


specific O-group: 4, 5 (B)

2


specific H: d, i

2

Shigella:

polyvalent flexneri, polyvalent boydii, polyvalent



dysenteriae, sonnei



specific: anti-Shigella dysenteriae 1 (Shiga)

1

Vibrio cholerae:

polyvalent 0:1

1


specific: ogawa, inaba

3

Neisseria meningitidis:

polyvalent and group-specific: A, B, C

3

Haemophilus influenzae:

type b

3