Collection,Transportation and Processing of Blood sample

Day 1

Gather blood and inoculate in culture media

Every time viable blood must be accumulated before antimicrobial treatment has started. When the patient has the recurring fever, accumulate the blood because the temperature begins to upward push. For other sufferers, collect the blood as quickly as possible after receiving the request. To culture the probabilities of setting apart a pathogen, it also includes endorsed that at least two specimens (gathered at special instances) ought to be cultured. A strict aseptic technique must be used to acquire the blood.

Choice of culture media

Decided media on for the culture of blood ought to be capable of offering the fastest growth and isolation of as wide more than a few pathogens as feasible. The following media are endorsed:

• Columbia agar and Columbia broth diphasic medium with added SPS (sodium polyacetal sulphonate), also known as Liquid. SPS prevents the blood from clotting, neutralizes complement and other antibacterial materials in sparkling blood, and has some neutralizing effect on polymyxin B, streptomycin, and gentamicin ought to these be present in the blood. SPS also allows a more volume of blood to be cultured without growing the volume of broth, i.e. as much as 50% of the full extent of a medium.
• Thioglycollate broth medium is suggested to isolate strict anaerobes ought to an anaerobic contamination be suspected. It consists of nutrient broth to which is delivered thioglycollate to offer the conditions necessary for the culture of anaerobes. Because SPS is inhibitory to anaerobic streptococci, it is not brought to this medium, consequently, an enough volume of broth ought to be used to prevent the blood from clotting and to dilute out the blood’s natural bactericidal substances. The blood ought to be diluted at the least 1 in 10 with broth.

Aseptic blood collection and dispensing method

Blood for culture ought to be accrued and dispensed with high-quality care to avoid contaminating the specimen and subculture medium.

1 .The use of a strain cuff, locate a suitable vein in the arm. Deflate the cuff while disinfecting the vene puncture site.

2 .Carrying gloves, thoroughly disinfect the venepuncture site as follows:

• –Using 70% ethanol, cleanse a place approximately 50 mm in diameter. allow to air-dry.
• The use of 2% tincture of iodine and a round action, swab the area beginning at the factor where the needle will input the vein.
• Permit the iodine to dry at the pores and skin for at least 1 minute.

3 .Raise lower back the tape or put off the protective cover from the pinnacle of the way of life bottle(s). Wipe the pinnacle of the bottle using an ethanol-ether swab.

4.Using a sterile syringe and needle, withdraw approximately 20 ml of blood from an adult or approximately 2 ml from a younger child. Collect 10–15 ml of blood.

5.Insert the needle thru the rubber liner of the bottle cap and dispense 10–12 ml of blood into the diphasic culture medium bottle containing 25 ml of broth . While also culturing for anaerobes, dispense approximately five ml of blood into the thioglycollate culture medium containing 50 ml of broth Dispense the ultimate approximately 2 ml of blood right into a tube or bottle containing ethylene diaminetetraacetic acid (EDTA).

6.Using a sparkling ethanol-ether swab, wipe the top of culture bottle and replace the tape or protecting cowl(s). At once,, blend the blood with the broth and mix the blood within the EDTA box.

Essential: The blood ought to now not be allowed to clot within the tradition media because any microorganism will grow to be trapped within the clot.

7.Truly label each bottle with the name and variety of the patient, and the date and time of collection.

8.As soon as feasible, incubate the inoculated media. guard the cultures from direct daylight until they're incubated. Diphasic medium Incubate at 35–37 °C for up to 7 days, examining and subculturing as defined later. A longer incubation period ought to be allowed whilst endocarditis

is suspected. while brucellosis is suspected, loosen the cap of the culture bottle (or insert a sterile needle into the cap) and incubate in a carbon dioxide enriched surroundings for as much as 4 weeks.Thioglycollate broth Incubate at 35–37 °C for up to two weeks, analyzing and subculturing as defined later.

Culture of blood from neonates

To lessen the hazard of infection, blood from neonates should be gathered from a peripheral vein, not from the umbilical vein. when most effective a small quantity of blood is obtained, inoculate it into a bottle of diphasic subculture medium. Organisms causing bacteraemia in younger kids are normally present in sufficient concentration to be detected in small volumes of blood (1–2 ml) of blood).Examine additionally a Gram-stained smear of the plasma-buffy coat layer, acquired by means of centrifuging anticoagulated capillary blood. it is often feasible to make a speedy analysis of bacteraemia in infants through this approach.

Observe the specimen microscopically

Centrifuge a sample of EDTA anticoagulated venous blood or heparinized capillary blood and make smears of the buffy coat layers. Stain as follows:

• Gram smear: To locate Gram positive and Gram negative microorganism, particularly while the patient is a little one or young baby.
• Ziehl-Neelsen smear: To hit upon AFB when the affected person has AIDS or suspected HIV ailment.
• Giemsa or speedy subject’s smear: To locate borreliae, or parasites inclusive of trypanosomes, malaria parasites, and microfilariae. permit the smears to air dry, repair with absolute methanol for two mins and stain by means of the correct staining approach.

Day 2 and Onwards

Observe and file the cultures Diphasic way of life (Columbia agar and broth) using a hand lens, observe two times daily (as much as 7 days or four weeks whilst brucellosis is suspected) for microbial growth, indicated by means of colonies developing on the agar slope, commonly starting at the agar-broth interface.

Colonial appearances

Colonies of staphylococci, S. Typhi, brucellae, and most coliforms can normally be visible without problems, while colonies of S. pneumoniae, Neisseria species, S. pyogenes, and Y. pestis are much less effortlessly visible. Pseudomonas and Proteus species produce a type of increase on the agar.

When growth is present:

• Culture on blood agar, chocolate agar, and MacConkey agar.
• Incubate the blood agar and MacConkey agar lates aerobically and the chocolate agar plate in a carbon dioxide atmosphere (candle jar).

Have a look at a Gram-stained smear of the colonies. relying on the bacteria visible, take a look at the colonies further (e.g. for coagulase, catalase, oxidase, urease, and motility).

• Whilst huge Gram wonderful rods such as C. perfringens are visible: culture also on lactose egg yolk milk agar and incubate the plate anaerobically .
• While motile, urease and oxidase terrible Gram-negative rods are remoted: culture the colonies on Kligler iron agar .
• while catalase positive Gram bad coccobacilli are isolated: Suspect Brucella species and send culture (securely) to a microbiology professional laboratory for identification. Mark the culture ‘excessive danger’.

Blind culture after in a single day incubation due to the fact a few organisms consisting of Neisseria species and S. pneumoniae may also develop without producing effortlessly visible colonies, it's miles really useful to study a toluidine blue stained smear and subculture onto agar plates even if no microbial increase is obvious after overnight incubation.

Important: constantly file right away an effective blood culture , and send a preliminary record of the stained smear and different useful check consequences. While Gram negative rods or staphylococci are visible in a Gram smear, set up suitable susceptibility exams.

Be aware: while no colony is visible on the slope of the diphasicculture , wash the broth over the slope before reincubating the subculture (do not allow the broth to circulate the neck of the bottle).

Thioglycollate broth culture

Have a look at each day (up to 14 days) for visible symptoms of bacterial increase together with turbidity above the crimson cell layer, colonies growing on the surface of the crimson cells (‘cotton balls’), hemolysis, fuel bubbles, and clots. maximum organisms (no longer just anaerobes) develop in thioglycollate broth. Whilst there are signs of bacterial increase, subculture the broth and take a look at a toluidine blue stained smear for bacteria.

Important: Whilst an affected person is seriously sick, culture the broth (even within the absence of seen bacterial growth) after in a single day incubation, after forty-eight hours, and two times weekly for up to two weeks.

Subculturing a blood culture broth

A strict aseptic technique has to be used to keep away from contaminating culture.

1.The use of an ethanol-ether swab, cleanse the pinnacle of the bottle. The use of a sterile needle and small syringe, insert the needle thru the rubber liner in the cap and withdraw about 1 ml of the broth culture.

2.Inoculate the broth on:

• Blood agar
• Chocolate (heated blood) agar
• MacConkey agar

Incubate the blood agar plate anaerobically for as much as forty-eight hours, the chocolate agar plate in a carbon dioxide atmosphere for as much as 48 hours, and the MacConkey agar plate aerobically in a single day.

3.Swab the pinnacle of the culture bottle and reincubate. These are usually organisms which are capable of developing at room temperature or below. They encompass coliforms, Achromobacter species, and pseudomonads and much less generally Yersinia species.

Possible Bacteria in Blood

Gram-positive

Staphylococcus aureus

Viridans streptococci

Streptococcus pneumonia

Streptococcus pyogenes

Enterococcus faecalis

Clostridium perfringens

Anaerobic streptococci

Gram-negative

Escherichia coli

Proteus species

Bacteroides fragilis

Neisseria meningitidis

Yersinia pestis

Klebsiella strains

Pseudomonas aeruginosa

Haemophilus influenza

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.