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close this bookBasic Laboratory Procedures in Clinical Bacteriology (WHO; 1991; 128 pages)
View the documentPreface
View the documentIntroduction
open this folder and view contentsQuality assurance in microbiology
close this folderPart I. Bacteriological investigations
open this folder and view contentsBlood
close this folderCerebrospinal fluid
View the documentIntroduction
View the documentCollection and transportation of specimens
View the documentMacroscopic inspection
View the documentMicroscopic examination
View the documentPreliminary identification
View the documentSusceptibility testing
open this folder and view contentsUrine
open this folder and view contentsStool
open this folder and view contentsLower respiratory tract infections
open this folder and view contentsUpper respiratory tract infections
open this folder and view contentsSexually transmitted diseases
open this folder and view contentsPurulent exudates, wounds, and abscesses
open this folder and view contentsAnaerobic bacteriology
open this folder and view contentsAntimicrobial susceptibility testing
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

Microscopic examination

Preparation of specimen

If, on gross examination, the CSF is purulent (very cloudy), it can be examined immediately without centrifugation. In all other cases, the CSF should be centrifuged in a sterile tube (preferably a 15-ml conical tube with screw-cap) at high speed for 5 - 10 minutes. Remove the supernatant using a sterile Pasteur pipette fitted with a rubber bulb, and transfer it to another tube for chemical and/or serological tests. Use the sediment for further microbiological tests.

Direct microscopy

Examine one drop of the sediment microscopically (X400), between slide and cover slip, for:


• leukocytes (polymorphonuclear neutrophils or lymphocytes),
• erythrocytes,
• bacteria,
• yeasts.

If the yeast-like fungus Cryptococcus neoformans is suspected, mix a loopful of the sediment with a loopful of India ink on a slide, place a cover slip on top, and examine microscopically for the typical, encapsulated, spherical, budding yeast forms.

In areas where African trypanosomiasis occurs, it will also be necessary to search carefully for actively motile, flagellated trypanosomes.

A rare and generally fatal type of meningitis is caused by free-living amoebae found in water (Naegleria fowleri) which enter through the nose and penetrate the central nervous system. They may be seen in the direct wet preparation as sluggishly motile amoebae about the size of neutrophilic leukocytes.

Gram-stained smears

As the causative agent of bacterial meningitis may often be observed in a Gram-stained smear, this examination is extremely important. Air-dry the smear, fix with gentle heat, and stain it by Gram’s method. Examine at x 1000 (oil-immersion) for at least 10 minutes, or until bacteria are found. Table 6 summarizes important diagnostic findings in different forms of meningitis.

Acid-fast stain (Ziehl-Neelsen)

Although its sensitivity is not high, examination of an acid-fast stained preparation of the sediment or of the fibrin web is indicated when tuberculous meningitis is suspected by the physician. Carefully examine the acid-fast stained preparation for at least 15 minutes. If the result is negative, the microscopic investigation should be repeated on a fresh specimen on the following day.


If bacteria have been seen in the Gram-stained smear, the appropriate culture media should be inoculated (see Table 7). If no organisms have been seen, or if the interpretation of the Gram smear is unclear, it is desirable to inoculate a full range of media, including a blood agar with a streak of Staphylococcus aureus to promote growth of H. influenzae. Blood agar and chocolate agar plates should be incubated at 35 °C in an atmosphere enriched with carbon dioxide. All media should be incubated for 3 days, with daily inspections.

Table 6. Cerebrospinal fluid findings associated with meningitis


Type of meningitis





Viral (“aseptic”)

Predominant white cell type

Segmented polymorphonuclear neutrophils

Mononuclear (young neutrophils)




Very low: 5-20 mg/100 ml

Low: 20-40 mg/100 ml

Low: 20-40 mg/100 ml

Normal: 65-70 mg/100 ml





Slightly elevated in early stage of infection

Stained smear

Bacteria usually seen (Gram)

Rarely positive (acid-fast)

Usually positive (India ink)


When tuberculous meningitis is suspected, at least three tubes of Löwenstein-Jensen medium should be inoculated with a drop of the sediment and incubated for 6 weeks. For the first 2-3 days the tubes should be incubated in a horizontal position with the screw-cap loosened half a turn. Tubes should be inspected for growth at weekly intervals. Smears from any suspicious growth should be prepared, preferably in a bacteriological safety cabinet, air-dried, heat-fixed, and stained by the Ziehl-Neelsen method. The presence of acid-fast rods is consistent with the diagnosis of tuberculosis. All isolates should be forwarded to a central laboratory for confirmation and for susceptibility testing.

When Cryptococcus neoformans is suspected, either from the India ink preparation or on clinical grounds, the sediment should be inoculated on two tubes of Sabouraud dextrose agar, and incubated at 35 °C for up to 1 month. C. neoformans also grows on the blood agar plate, which should be incubated at 35 °C for I week, if indicated.

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