Culture of C.S.F Sample

Cell count

A white cellular count with an indication whether the cells are pus cells or lymphocytes is needed whilst the c.s.f. seems slightly cloudy or clean or whilst the Gram smear does no longer suggest pyogenic bacterial meningitis.

Approach

To pick out whether or not white cells within the c.s.f. are polymorphonuclear neutrophils (pus cells) or lymphocytes, dilute the c.s.f. in a fluid which stains the cells. Isotonic zero.1% toluidine blue is suggested as it stains lymphocytes and the nuclei of pus cells blue. C. neoformans yeast cells stain red. red cells stay unstained. The motility of trypanosomes isn't affected by the dye. while toluidine blue is unavailable, isotonic methylene blue may be used on the way to additionally stain the nuclei of leucocytes. If favored, leucocytes may be differentiated by way of analyzing a Leishman, Giemsa or speedy discipline’s stained smear (sediment from centrifuged c.s.f.) after counting the cells.

1. Mix the c.s.f. (pattern No. 2 uncentrifuged c.s.f.). Dilute the fluid 1 in 2, i.e. blend 1 drop of c.s.f. with one drop of toluidine blue diluting .

2 .Bring together a modified Fuchs-Rosenthal dominated counting chamber,making sure the chamber and cowl glass are absolutely clean.

3.The use of a fine bore Pasteur pipette or capillary tube cautiously fills the counting chamber with the well-combined diluted c.s.f. The fluid must now not overflow into the channels on each aspect of the chamber.

4.Wait approximately 2 mins for the cells to settle. Count the cells microscopically.

5.Awareness the cells and rulings the usage of the 10_ goal with the condenser iris closed sufficiently to provide the correct assessment. earlier than beginning the depend on without, use the 40_ objective to test that the cells are white cells and not red cells (unstained small without the nucleus) and word whether or not the white cells are in particular polymorphonuclear neutrophils (with the lobed nucleus) or lymphocytes. If a mixture of each, estimate approximately the proportion of each kind of cell. Note: When purple cells are visible, mention this within the document. when many red cells are present, the c.s.f. is unsuitable for WBC cell counting.

6.Depend on the cells in five of the massive squares.

Biochemical checking out OF C.S.F.

Biochemical c.s.f. exams which may be required consist of the dimension of protein and glucose. Be aware: When the Gram smear shows organisms and pus cells, little extra records will be furnished by means of checking out for protein and glucose. When however no bacteria are seen in the Gram smear and the cellular count number is raised, trying out for protein and glucose can help to differentiate those situations in which lymphocytes are observed in c.s.f., e.g. viral meningitis (barely raised protein, everyday glucose) from tuberculous meningitis (high protein, low glucose).

Measurement of c.s.f. glucose

Glucose needs to be measured inside 20 minutes of the c.s.f. being withdrawn in any other case a falsely low-end result might be acquired because of glycolysis. Use the supernatant fluid from centrifuged c.s.f. or uncentrifuged c.s.f. if the pattern seems clear. Glucose may be measured in c.s.f. using a colorimetric technique or a less complicated semiquantitative technique the use of Benedict’s reagent. Raised c.s.f. glucose: takes place while the blood glucose stage is raised (hyperglycaemia) and from time to time with encephalitis. Low c.s.f. glucose: The c.s.f. glucose concentration is decreased in maximum forms of meningitis, besides viral meningitis. In pyogenic bacterial meningitis, it's miles markedly decreased and might even be undetectable.

Measurement of c.s.f. overall protein and globulin test

Use the supernatant fluid from centrifuged c.s.f. or uncentrifuged c.s.f. When the sample appears clear. The overall protein may be measured in c.s.f. the usage of a colorimetric technique or a visible comparative technique. Pandy’s test is a screening test which detects rises in c.s.f. globulin. It's far of fee whilst it is not feasible to degree c.s.f. overall protein. Ordinary c.s.f. protein: overall c.s.f. protein is generally 0.15–0.40 g/l (15–forty mg%). The range for ventricular fluid is slightly lower. Values up to one.zero g/l (a hundred mg%) are normal for new child infants. most effective lines of globulin are observed in ordinary c.s.f., insufficient to supply a high-quality Pandy’s test. Expanded c.s.f. general protein with tremendous Pandy’s Test: happens in all sorts of meningitis, in amoebic and trypanosomiasis meningoencephalitis, cerebral malaria, mind tumours, cerebral harm, spinal cord compression, poliomyelitis, the Guillain-Barré syndrome (frequently the simplest abnormality), and polyneuritis. Increases in c.s.f. protein additionally occur in illnesses which motive adjustments in plasma proteins together with myelomatosis. Whilst the full protein exceeds 2.0 g/l (2 hundred mg%), the fibrinogen level is usually expanded sufficiently to purpose the c.s.f. to clot. this will occur in extreme pyogenic meningitis, spinal block, or following haemorrhage.

.More Investigations

Ziehl-Neelsen smear whilst tuberculous meningitis is suspected

Study a Ziehl-Neelsen stained c.s.f. smear for acid-fast bacilli (AFB) whilst tuberculous meningitis is clinically suspected or the c.s.f. consists of lymphocytes and the glucose attention are low and the protein raised. AFB, however, are hard to discover in c.s.f. the subsequent method increases the chances of locating the microorganism:

1. Centrifuge the c.s.f. at excessive pace for 20–half-hour. put off the supernatant fluid and mix the sediment. Transfer numerous drops of the sediment to a slide, permitting each drop to dry earlier than adding the subsequent.

2 .Repair the dry preparation with methanol and stain via the Ziehl-Neelsen technique .

3.Look at the smear first with 40_ objective to peer the distribution of material after which with the 100_ goal to come across the AFB. observe the whole practise. the appearance of M. tuberculosis in a Ziehl-Neelsen stained smear.

India ink Preparation when cryptococcal meningitis is suspected

When cryptococcal meningitis is clinically suspected, e.g. affected the person with HIV disorder, or whilst yeast cells are detected when acting a cell depend on or inspecting a Gram smear, examine an India ink instruction or a wet coaching by means of dark-field microscopy for encapsulated yeasts.

1.Centrifuge the c.s.f. for 5–10 mins. cast off the supernatant fluid and blend the sediment.

2.transfer a drop of the sediment to a slide, cover with a cover glass and examine by dark-field microscopy . or add a drop of India ink (Pelikan black drawing ink is suitable*), blend and cover with a cover glass. Note: Do now not make the preparation too thick in any other case the cells and pills will not be seen.

3.Observe the training the use of the 40_ objective. Search for oval or round cells, some showing budding, irregular in length, measuring 2–10 _m in diameter and surrounded by way of a large unstained capsule.Very sometimes pills are absent. Important: while encapsulated yeasts are detected in c.s.f., a presumptive diagnosis of cryptococcal meningitis can be made. The medical officer attending the affected person should be notified at once. further information on C. neoformans may be discovered.

Wet preparation and Giemsa smear when trypanosomiasis meningoencephalitis is Suspected

Clean c.s.f. is needed to stumble on trypanosomes. approximately 15 mins after the fluid is withdrawn, the trypanosomes begin to lose their motility and are unexpectedly lyzed. The trypanosomes are normally few and therefore a careful seek of a moist coaching is needed to locate the motile flagellates.

1.Centrifuge the c.s.f. at about one thousand g for 10 mins. put off the supernatant fluid and blend the sediment.

2.switch a drop of sediment to a slide and cowl with a cowl glass. have a look at for motile trypanosomes the use of the 40_ objective with the condenser iris closed sufficiently to offer excellent evaluation. alternatively, have a look at the preparation by using darkfield microscopy . Take a look at a moist training for motile amoebae whilst primary amoebic meningoencephalitis is clinically suspected (uncommon situation due to N. fowleri) or the c.s.f. Incorporates pus cells with raised protein and low glucose, but no microorganism are visible within the Gram smear. Red cells will also be present.

3..Transfer a drop of uncentrifuged purulent c.s.f. or a drop of sediment from a centrifuged specimen to a slide and cowl with a cowl glass.

4..Observe the guidance the use of the 10_ and 40_ targets, with the condenser closed sufficiently to present exact evaluation. look for small, clean, motile, elongated forms most of the pus cells. Use the 40_ objective to perceive the amoebae (even if not first seen using the 10_ objective, usually take a look at the education with the 40_ objective). The amoebae frequently comprise vacuoles however not pink cells.

Day 2 and Onwards

Take a look at and report the cultures Chocolate agar and blood agar cultures appearance specifically for colonies that would be:

• Neisseria meningitidis (growing on chocolate agar and blood agar, oxidase superb .
• Streptococcus pneumoniae (touchy to optochin,
• Haemophilus influenzae (growing simplest on chocolate agar.
• Cryptococcus neoformans (Gram stain the colonies).

MacConkey agar culture

Appearance especially for colonies that would be:

• Escherichia coli or another coliform.
• Streptococcus agalactiae.
• Listeria monocytogenes.
• Other bacteria that reason neonatal meningitis

Antimicrobial susceptibility testing

Test isolates of S. pneumoniae for susceptibility to chloramphenicol and penicillin (use 1_g oxacillin disc). check H. influenzae for beta-lactamase manufacturing .and susceptibility to chloramphenicol (using chocolate agar). carry out susceptibility checking out on Gram terrible rods as indicated.

Possible pathogens

Gram-positive micro organism

• Streptococcus pneumoniae
• Streptococcus agalactiae
• Listeria monocytogenes
• Streptococcus is

Gram-negative organisms

• Neisseria meningitidis
• Haemophilus
• influenzae type b
• Escherichia coli
• Pseudomonas
• aeruginosa
• Proteus species
• Salmonella serovars
• Flavobacterium
• Meningosepticum

References:

D greenwood, Slack RCB and J Peutherer.Medical microbiology.2001.

JG College, AG Fraser and BP Marmion.Practical Medical microbiology.Fourteenth Edition. Churchill Livingstone, 1996.

JP Micheal, ECS Chan and NR Krieg.Microbiology.Fifth Edition. Delhi: Mcgraw-hill, 1993.

M Cheesbrugh.Medical laboratory manual for tropical countries.London, 2007.