Microbiologylog

WBCs and RBCs count in sterile body fluids (such as CSF)

The quantity of white blood cells and red blood cells in sterile body fluids (such as CSF) can be obtained by performing a cell count on the uncentrifuged specimen, preferably the last specimen taken, using a modified mirrored Fuchs Rosenthal counting chamber. The cell count of sterile body fluids specimen such as cerebrospinal fluids (CSF) usually gives important clues of presence of infection or not and if present, the infecting organism. Sequential specimens 1 to 4 are usually obtained from one lumbar puncture and specimen 1 is sent to Clinical Biochemistry laboratory while 2 – 4 are sent to Microbiology laboratory. Red cell count is done on the entire 3 Specimen sent to Microbiology laboratory and both RBC and WBC count is performed on specimen 4. Uniform blood staining of all samples suggests previous haemorrhage into the subarachnoid space, whereas reducing counts in sequentially obtained samples suggest bleeding induced by the tap procedure. CSF obtained more than 12 hours post intra-cranial haemorrhage may show raised WBC counts of up to 500 x 10⁶/l as a result of an inflammatory response.

The procedure for performing a cell count starts with drawing a line with a chinagraph pencil on the external supports of the clean and dry counting chamber and then gently pushes the cover glass onto the counting chamber from the front. The formation of interference lines (Newton rings) between the external support and the cover glass shows that the cover glass is correctly positioned. Fill the counting chamber with the specimen using a fine tip sterile pipette and allows to settle for 5 minutes. Observe under x10 objective lens microscope to focus and the x10 or x40 to count cells.

The modified Fuchs Rosenthal counting chamber has nine (9) large triple lined squares; each divided in 16 small squares and has a depth of 0.2 mm. as shown in Figure below. Each large square is 1 mm²; therefore 5 large triple lined squares are counted to get the count /mm³. The four corner squares and the middle are also counted. If cells are lying on the triple lines between squares count only the cells lying on the inner two lines of the top and the left side lines.

The ratio of WBC and RBC in a normal blood in WBC1-2: 1000 RBC and under normal conditions the number of white cells in a CSF is <5/mm³ in children and <20/mm³ in adults. In some situations where the sample is turbid or blood stained, with a high expectation of RBC count of >200 mm³, dilution is often performed in sterile saline before loading on the counting chamber. The cell count result must then be multiplied by the dilution factor.

Normal CSF values

Normal CSF values Neonates Adults

Leucocytes Neonates 0-30 cells / cu mm ( x 10⁶/l)

1-4yr old 0-20 cells / cu mm ( x 10⁶/l)

5yr-puberty 0-10 cells / cu mm ( x 10⁶/l)

Adults 0-5 cells / cu mm ( x 10⁶/l)

Erythrocytes Newborn 0-675 cells / cu mm ( x 10⁶/l)

Adults 0-10 cells / cu mm ( x 10⁶/l)

The counting chamber grid

Processing of NEQAS Corynebacterium diphtheriae specimen

I reconstituted the lyophilised material/specimen from NEQAS with 1ml of nutrient broth, gently mixed and left for 5 minutes. This is then plated on culture media which includes Hoyles Tellurite agar and blood agar. Hoyles Tellurite agar is incubated in air for 48 hours at 35-37^oC while blood agar are incubated anaerobically for 24-48 hours at 35-37^oC. The culture plates where observed at 24 and 48 hours for growth with sufficient growth observed at 24 hours. They were grey/black colonies on the Hoyle’s tellurite agar while on blood agar, the colonies appear grey and granular with irregular edges and small zone of haemolysis. Gram stain was performed on the isolates and they show Gram positive rods with some of them in pairs, lying on each, slightly curved, clubbed end and resembling Chinese letters.

Biochemical test (catalase test) was performed following culture growth and it was positive. The isolate was then identified using Biomerieux VMS (MALDI-TOF) as C. diphtheria. For patient samples, as soon as the isolate is identified as C. diphtheria the consultant microbiologist will be informed and chocolate agar slopes inoculated which will be sent to our reference laboratory for confirmation, biotyping and toxigenicity. Health and safety procedures should be strictly followed when processing a suspected diphtheria specimen including the use of Class I safety cabinet and personal protective equipment.

It is also important to establish whether the isolated C. diphtheria is toxigenic or not. This is done with the use of ELEK double diffusion precipitin method at our reference laboratory at Health Protection Agency, Respiratory and systemic infections laboratory (RSIL), Streptococcus and Diphtheria Unit, Colindale, London. The test is performed with the use of a filter paper strip impregnated with diphtheria antitoxin which is buried just beneath the surface of a special agar plate before the agar solidifies. Positive and negative known toxigenic strains plus the strains to be tested are streaked on the agar surface in a line across the plate and at a right angle to the antitoxin paper strip. The plate is then incubated for 24 hours at 35-37^oC. Transmitted light is used to examine the plate after incubation for the presence of fine precipitin lines at 45 degrees to the streak. Toxin producing strains reacts with the antitoxin with the formation of precipitin lines.

Diphtheria is an acute contagious disease caused by Corynebacterium diphtheriae usually affecting the upper respiratory tract mucosa. It is characterised by the formation of a fibrinous pseudomembrane. It also causes damage to the myocardial and neural tissue caused by the action of a potent exotoxin. The organism grows in the upper respiratory tract where it is absorbed into the mucous membrane producing exotoxin known as diphtheria toxin which causes destruction of the epithelium and a superficial inflammatory response. Once the exotoxin is absorbed into the mucous membrane, they can be carried in the circulation into distant organs such as heart. There are four recognised biotypes of C. diphtheria which include mitis, gravis, intermedius and belfanti with gravis known to produce the most exotoxin and thus more pathogenic that the rest.

Salmonella spp. from Blood culture

Salmonella are predominantly motile (most species except Salmonella pullorum-gallinarum are motile with peritrichous flagella), non-spore forming, Gram negative rod bacteria that belongs to Enterobacteriaceae family. They are divided into two species – Salmonella bongori (previously serotype V) and Salmonella enterica (Salmonella enterica is subdivided into subspecies – enteric (serotype I), salamae (serotype II), arizonae (serotype IIIa), diarizonae (serotype IIIb), houtenae (serotype IV) and indica (serotype VI), and over 2500 serovars. The serovars are defined based on the somatic O (Lipopolysaccharide) and flagellar H antigens (The Kauffman-White classification).

The blood culture aerobic and anaerobic bottle flagged up positive after 8 hours incubation in the BD Bactec FX Blood culture analyser and upon Gram stain shows Gram negative rods. Routine blood culture plates where inoculated according the Laboratory SOP - Blood agar, chocolate agar, UriSelect chromogenic agar and FAA AN anaerobic plates. All culture plates were purchased from E & O Labs (http://www.eolabs.com/). After 18-48 hours incubation at appropriate temperatures and condition, there was growth on all plates. The Salmonella isolate on culture plates are shown below.

Culture medium	Incubation condition	Incubation Time	Appearance
Blood agar	CO₂at 37^OC	18 – 48 hours	moist, grey, round non-haemolytic colony
Chocolate agar	CO₂at 37^OC	18 – 48 hours	moist, grey, round colony
UriSelect chromogenic agar	Air at 37^OC	18 – 24 hours	moist, white/cream, round colony

This isolate was then identified using Biomerieux Vitek MS (MALDI-TOF) as Salmonella group. Purity plates were done on CLED plates and used for agglutination test after 24 hours incubation at the appropriate temperature condition. Salmonella isolates are used for serological confirmation using the agglutination test Polyvalent O and Polyvalent H specific antisera. This is an easy, quick and straightforward method. Specialist reference microbiology laboratories use more advanced and automated methods such as PCR technique.

The Kaufmann-White scheme is a system complex serotyping scheme that classifies Salmonella into serotypes based on their surface antigens. Specific antisera are raised against three antigenic sources; the ‘O’ polysaccharide component of the bacterial cell wall, the ‘H’ flagellar antigens present in most strains and the ‘Vi’ virulence/capsular antigen present mainly, but not exclusively, in S.typhi. It was further found that each strain could possess several ‘O’ determinants and so could be attributed to a particular ‘O’ group. Additional subdivision could also be made on the basis of flagella ‘H’ antigens as Salmonella exhibit phase variation between motile (phase 1) and non-motile (phase 2) phenotypes. The Kaufmann-White scheme was first published in 1934 listing 44 serotypes, but now contains serotypes for more than 2500 Salmonella spp and is used both in routine and specialised laboratories for the identification of Salmonella species. Examples of the serotypes can be seen on the table below

Serotypes	‘O’ Antigens	‘H’ Antigens
Serotypes	‘O’ Antigens	Phase 1	Phase 2
Salmonella typhi	9,12,Vi	d	None
Salmonella paratyphi A	1,2,12	a	None
Salmonella paratyphi A var. Durazzo	2,12	a	None
Salmonella paratyphi B	1,4,5,12	b	1,2
Salmonella paratyphi B var. Odense	1,4,12	b	1,2
Salmonella enteritidis	1,9,12	g,m	None
Salmonella typhimurium	1,4,5,12	i	1,2