1. Blood culture lecture NO 5&6
Dalia Kamal Eldien Mohammed

2. introduction
 The bloodstream is usually a sterile environment, the isolation of any organism from a blood culture must be considered significant and correlated with the clinical picture.
A blood culture is a fairly routine test that checks for bacteria, yeast, and other microorganisms in the blood.
Blood cultures are collected from patients with suspected sepsis or bacteremia.

3. Bacteraemia
Is the presence of bacteria in the blood
It is usually pathological, although transitory asymptomatic bacteraemia can occur during the course of many infections and following surgical procedures.
Bacteraemia occurs in diseases such as typhoid fever, brucellosis, leptospirosis and endocarditis.

4. Septicemia
This is a clinical term used to describe severe life-threatening bacteraemia in which multiplying bacteria release toxins into the blood stream and trigger the production of cytokines, causing fever, chills, toxicity, tissue anoxia, reduced blood pressure, and collapse.
Septic shock is usually a complication of septicemia with Gram negative bacilli.

5. Indication of blood culture
Endocarditis
Suspected deep fungal infection, such as histoplasmosis and blastomycosis.
Suspected mycobacteremia caused by Mycobacterium avium-, particularly in HIV patients with CD4 counts <50. 
Suspected disseminated gonococcal infection.
Suspected candidemia or disseminated cryptococcosis
Suspected Malassezia furfur infection, an agent of catheter-associated infection in patients receiving intravenous lipid.
patients with fever and leukocytosis or leukopenia, However, a normal white blood cell count does not rule out bacteremia

6. Symptoms Chills Confusion Decreased urine Fever Nausea Rapid breathing
Rapid heartbeat

7. Why we do  blood culture?
Find a bacterial infection that has spread into the blood, such as meningitis, osteomyelitis, pneumonia, a kidney infection, or sepsis.
Find a fungal infection, such as yeast, in the blood.
Check for endocarditis, which is an infection of the heart valves  .
Find the best antibiotics to kill the bacteria or fungi.
Find the cause of an unexplained fever or shock or a person becoming extremely ill.

8. TIMING
Blood cultures should be drawn prior to the institution of antibiotics whenever possible.
SITE OF BLOOD CULTURE
Blood should be obtained from peripheral venous or arterial sites.
Obtaining blood cultures from central venous catheters, arterial lines and inguinal vessels increases the likelihood of obtaining a false positive blood culture. 
The practice of drawing blood for culture from catheters or the groin should never be performed when a peripheral site is available.

9. VOLUME OF BLOOD PER SET
There is a direct relationship between the volume of blood obtained and the yield of a blood culture set.
Organisms causing bacteraemia in young children are usually present in sufficient concentration to be detected in small volumes of blood (1–2 ml) of blood).
A minimum of 10 ml (and preferably 20 ml) of blood should be obtained from adults, and ml (and preferably more) from infants and children

10. NUMBER OF SETS OF BLOOD CULTURES
Single sets should not be used to evaluate any patient with suspected bacteremia or candidemia, a single blood culture may miss intermittently occurring bacteremia and make it difficult to interpret the clinical significance of certain isolated organisms.
The optimal yield is obtained with three or four sets of blood cultures, at least two sets.
No more than four blood cultures should be obtained for any given 24 hour period.

11. Possible pathogens isolated from blood cultures
Gram positive Gram negative
Staphylococcus aureus Salmonella Typhi
Viridans streptococci Other Salmonella serovars
Streptococcus pneumoniae Brucella species
Streptococcus pyogenes Haemophilus influenzae
Enterococcus faecalis Pseudomonas aeruginosa
Clostridium perfringens Klebsiella strains
Anaerobic streptococci Escherichia coli
Proteus species
Bacteroides fragilis
Neisseria meningitidis
Yersinia pestis

12. Possible pathogens isolated from blood cultures
Also Mycobacterium tuberculosis (HIV-associated tuberculosis
Leptospira species
Borrelia species
Rickettsiae
Bartonella bacilliformis.
FUNGI
Candidaalbicans and other yeasts, e.g. Cryptococcus neoformans, and occasionally Histoplasma capsulatum and other fungi that cause systemic mycoses.

13. Question
Enumerate the blood commensals bacteria?????

14. Answer Blood does not have a normal microbial flora.
Common skin contaminants include
Coagulase negative Staphylococci
viridans streptococci
Micrococci
Corynebacterium species.

15. Laboratory diagnosis Specimen collection
SKIN PREPARATION
Clean the vein puncture site with 70% isopropyl alcohol an area about 50 mm in diameter. Allow to air-dry.
sterilize with a 1 to 10% povidone-iodine solution or chlorhexidine- gluconate(for infants), in a circular action, swab the area beginning at the point where the needle will enter the vein.
Allow the site to air dry for at least 1minute.

16. Clean with alcohol swab

17. sterilize with a 1 to 10% povidone-iodine

18. PREPARE BACTEC VIALS
label culture vial.
Lift back the tape or remove the protective cover from the top of the culture bottle(s).
Wipe the top of the bottle using an ethanol-ether swab, and allow to dry
At least 2 sets of blood cultures should be collected from a patient with suspected bacteremia .

19. blood should be collected before antimicrobial treatment has started.
When the patient has recurring fever, collect the blood as the temperature begins to rise.
The ideal is to collect blood at time intervals ranging from one to several hours
A minimum of 10 ml (and preferably 20 ml) of blood should be obtained from adults, and ml (and preferably more) from infants and children

20. SPECIMEN LABELING
1. Each vial should be labeled with the appropriate patient information:
Patient’s name
Hospital number (Patient ID)
Patient’s location (room and bed )
Date and time of collection
Collector’s initials
Site of venipuncture
Or other information as per facility
2. Each request slip should also have all the information above.

21. Culture:
Media selected for the blood culture should be capable of providing the fastest growth and isolation of as wide a range of pathogens as possible
Using a fresh ethanol-ether swab, wipe the top of each culture bottle and replace the tape or protective cover(s).
Without delay, mix the blood with the broth, The blood must not be allowed to clot in the culture media because any bacteria will become trapped in the clot.
As soon as possible, incubate the inoculated media.
Protect the cultures from direct sunlight until they are incubated
the common types are:

22. Columbia agar and Columbia broth diphasic medium with added SPS (sodium polyanethol sulphonate), also known as Liquoid. SPS prevents the blood from clotting, neutralizes complement and other antibacterial substances in fresh blood, and has some neutralizing effect on polymyxin B, streptomycin, and gentamicin
Thioglycollate broth medium is recommended to
isolate strict anaerobes
III. Mycobacterial blood culture bottles contain broth suitable for the isolation of mycobacteria

23. Prior inspection of culture media bottles
Do not use a bottle of culture medium if it shows signs of contamination, i.e. broth appears turbid.
Do not use a bottle of thioglycollate broth if it appears oxidized, i.e. more than a third of the top of the medium appears pink when the indicator in the medium is resazurin, or more than 20 mm down from the surface of the medium appears green-blue when the indicator is methylene blue.
When oxidation has occurred, the medium must be reduced by steaming before it is used.

24. Diphasic media

25. Inoculated culture media

26. Direct Microscopically Exam
Centrifuge a sample of EDTA anti coagulated venous blood or heparinized capillary blood and make smears of the Buffy coat layers, It is often possible to make a rapid diagnosis of bacteraemia in infants by this method.
Stain as follows:
Gram stain
ZN To detect AFB when the patient has AIDS o suspected HIV disease
Giemsa or rapid Field’s, To detect borreliae, or parasites such as trypanosomes, malaria parasites, and microfilariae.

27. Incubation For the Diphasic medium
Incubate at 35–37 °C for up to 7 days, examining and subculturing as described later.
A longer incubation period should be allowed when endocarditis is suspected.
When brucellosis is suspected, loosen the cap of the culture bottle (or insert a sterile needle in the cap) and incubate in a carbon dioxide enriched atmosphere for up to 4 weeks.
For theThioglycollate broth
Incubate at 35–37 °C for up to 2 weeks

28. Examine and report the cultures
Diphasic culture (Columbia agar and broth) Using a hand lens, examine twice daily (up to 7 days or 4 weeks when brucellosis is suspected) for microbial growth, indicated by colonies growing on the agar slope, usually beginning at the agar-broth interface.
Important: Always report immediately a positive blood culture, and send to physician

29. When growth is present:
Subculture on blood agar, chocolate agar, and MacConkey
Incubate the blood agar and MacConkey aerobically and the chocolate agar plate in a carbon dioxide atmosphere (candle jar).
Examine a Gram stained smear of the colonies.
Depending on the bacteria seen, test the colonies further (e.g. for coagulase, catalase, oxidase, urease, and motility).

30. Thioglycollate broth culture
Examine daily (up to 14 days) for visible signs of bacterial growth such as turbidity above the red cell layer, colonies growing on the surface of the red cells (‘cotton balls’), haemolysis, gas bubbles, and clots.
Most organisms (not just anaerobes) grow in thioglycollate broth.
When there are signs of bacterial growth, subculture

31. Technique of subculture from broth media
A strict aseptic technique must be used to avoid contaminating the culture.
Using an ethanol-ether swab, cleanse the top of the bottle.
Using a sterile needle and small syringe, insert the needle through the rubber liner in the cap, and withdraw about 1 ml of the broth culture.
Inoculate the broth on:
– Blood agar
– Chocolate (heated blood) agar
– MacConkey agar
Incubate the blood agar plate anaerobically for up to 48 hours, the chocolate agar plate in a carbon dioxide atmosphere for up to 48 hours, and the MacConkey agar plate aerobically overnight.

32. subculture

33. Identify??

37. Contamination of blood cultures
Contamination can occur when an aseptic technique is not used at the time the blood is collected or at a later stage in the laboratory when subculturing.
Frequent contaminants of blood cultures include commensal staphylococci, micrococcus, and diphtheroids or contaminants from the environment such as species of Bacillus
Occasionally in immunocompromised patients, organisms usually considered ‘contaminants’ may be pathogenic, especially fungi.
Contamination is indicated when an organism is recovered from only one bottle when it should have grown in both thioglycollate broth and the diphasic culture medium or when a mixed microbial flora is isolated.

38. AUTOMATED SYSTEMS
Many techniques have been developed to reduce the technician manipulation time, to detect positive blood cultures more reliably, to reduce the contamination rat and to identify positive cultures more rapidly than manual systems.
The detection of increased C02 concentrations due to microbial growth is widely applied with different techniques.
With the development of infrared spectrophotometric C02 detection, a new generation of blood-culture instruments was introduced in the 1980s by Becton Dickinson Diagnostic Instrument Systems

39. The BACTEC 9120 and 9240 systems
The BACTEC blood culture system is a fully automated microbiology growth and detection system designed to detect microbial growth from blood specimens. The BACTEC FX and the BACTEC 9000 family of continuous monitoring blood culturing instruments offering performance, safety, reliability, ease of use, media quality and service. 
The Vital automated blood-culture system
BioArgos system is a fully automated blood culture system that detects carbon dioxide production by infrared spectroscopy through a glass bottle

40. Automated system

41. Reference
Murray PR, Traynor P, Hopson D. Critical assessment of blood culture techniques: analysis of recovery of obligate and facultative anaerobes, strict aerobic bacteria, and fungi in aerobic and anaerobic blood culture bottles. J Clin Microbiol 1992;30: