Blood culture: Collection and Transport of Body Fluids

Collection and Transport of Blood and Body

  Fluids for Culture

 

GENERAL CONSIDERATIONS:

The proper collection of sample is the most important step in the ultimate confirmation that a microorganism is responsible for the infectious disease process.

Failure to isolate the causative organism in an infectious process is not necessarily the fault of inadequate cultural methods; it frequently results from faulty collecting or transport techniques.

So, following are the general considerations recommended regarding the collection and transport of material for culture

• Whenever possible specimen should be collected before antimicrobial agents have been administered.

• The material should be collected where the suspected organisms is most likely to be found, with as little external contamination is possible.

• Another factor that contributes in successful isolation of organism is the stage of the disease at which the specimen is collected for culture.

• Specimens should be of a sufficient quantity to permit complete examination.

• The specimen should be placed in a sterile leak-proof container to prevent hazard to the handling staff.

• Provision should be made for the prompt delivery of specimen to the laboratory.

• One important thing is that clinicians should give sufficient clinical information to guide the microbiologist in selection of suitable media and appropriate technique.

• The clinicians must appreciate the limitations and potentials of the bacteriology laboratory and realize that a negative result does not necessarily invalidate the diagnosis.

• Laboratory personnel should reject specimens not obtained in a proper manner.

• Each specimen must be clearly labelled with the date and time of collection, patient’s name, number, ward or health centre.

• Each specimen must be accompanied by a request form which includes the following:

• Patient’s name, age, number and ward or health centre with name of consulting doctor.
• Type of specimen and date and time of its collection.
• Clinical diagnosis with patient’s history.
• Patient`s immune status.
• Any antimicrobial treatment that may be started at home or in the hospital.
• Specimens containing dangerous pathogens should be labelled as HIGH RISK and immediately after collection they should be sealed inside plastic bag or in a container with a tight — fitting lid.

• HIGH RISK specimens include:
• Sputum likely to contain M. tuberculosis.
• Faecal specimen that may contain V.cholera or S. typhi.
• Specimens from patients with highly contagious infections like HIV, Hepatitis, Viral haemorrhagic fever or plague.

 

BLOOD CULTURE

Blood culture is the most useful tool to detect

microorganism from cases of pyrexia of unknown

 origin.

 

DEFINITIONS:

• Bacteraemia: The presence of bacteria in blood is called bacteraemia. 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.

• Septicaemia: The term septicaemia refers to a severe and often fatal infection of the blood in which bacteria multiply and release toxins into the blood stream. The commonest portals of entry for bacteraemia/septicaemia are the genitourinary tract, respiratory tract, abscesses, surgical wound infections, biliary tract and miscellaneous sites.

 

COMMON PATHOGENS CAUSING SEPTICEMIA

 

BACTERIA	GRAM POSITIVE: Staphylococcus aureus, Viridans streptococci, Streptococcus pneumonia, Streptococcus pyogenes, Enterococcus faecalis, Clostridium perfringens, Anaerobic streptococci.
	GRAM NEGATIVE: Salmonella typhi, other Salmonella species, Brucella species, Haemophilus influenza, Pseudomonas aeruginosa, Klebsiella species, Escherichia coli, Proteus species, Bacteroides fragilis, Neisseria meningitides, Yersinia pestis
OTHER BACTERIA	Mycobacterium tuberculosis, Leptospira species, Borrelia species, Rickettsiae and Bartonella bacilliformis
FUNGI	Candida albicans and other yeasts and occasionally Histoplasma capsulatum and other fungi that cause systemic mycoses

 

INDICATIONS FOR BLOOD CULTURE

• Septicaemia, an often life — threatening microbial invasion from an infected focus accompanied by increase in temperature, increase in pulse rate or chills followed by fever.

• Bacteraemia, which accompanies chronic infections such as disseminated gonococcal disease or severe infections exemplified by meningitis, pneumonia, or deep seated abscesses.

• Intravascular infections such as endocarditis, thrombosed blood vessels or those due to intravenous catheters.

• Bacteraemia of multisystem infections such as enteric fever, leptospirosis, brucellosis.

• Bacteremia secondary to traumatic insults and instrumentation such as puncture wounds, urinary tract catheterization, contaminated intravenous medication.

• Patients having fever of unknown origin (FUO).

 

TIMINGS FOR COLLECTION OF BLOOD FOR CULTURE

• In conditions like undrained abscesses, instrumentation of contaminated mucosal surfaces, manipulation of infected tissues, bacteria are transiently present in the blood stream.

• During early stages of typhoid fever, brucellosis, bacteria are continuously present in the blood stream.

• While in bacterial endocarditis, septic shock, organisms are released into the blood stream at fairly constant rate making timing of cultures unimportant.

• So blood should be collected at the time of the patient’s temperature beginning to rise. (Except bacterial endocarditis where blood can be collected at any time).

 

MEDIA FOR BLOOD CULTURE

• Blood culture media should contain a nutrient broth and an anticoagulant.
• (i) Tryptic soya broth.
• (ii) Brain heart infusion broth.
• (iii) Thioghycollate broth.

 

COLLECTION OF BLOOD

• Blood is collected by proper phlebotomy. Skin should be cleaned properly before collecting blood, this is necessary to reduce risk of introducing contaminants into blood culture media. 

• It is less desirable to draw blood through a vascular shunt or catheter since these prosthetic devices are difficult to decontaminate completely.

• It is also recommended to draw blood below an existing intravenous line if possible, since, blood above the line will be diluted with the fluid being infused.

• If the blood is not being inoculated directly into broth media, it must be transported with an anticoagulant sodium polyanethol sulfonate (SPS, Liquoid). 

• SPS is also anticomplementary, antiphagocytic and interferes with the activity of some antimicrobial agents, notably aminoglycosides. Heparin, EDTA and Citrate have been found to be inhibitory to a number of organisms.

 

Method:

Total aseptic precautions, just like minor surgical preparations are required.

(i) Using a pressure cuff, locate a suitable vein in the arm.

(ii) Cleanse thoroughly the skin over the vein using tincture of iodine followed by ethanol/ether.

(iii) Remove the protective cap from the top of the culture bottle and cleanse the top of each bottle using an ethanol/ether swab.

(iv) Using a sterile syringe and size 2l/suitable gauge needle withdraw 10 ml of blood.

(v) With care, remove the needle from the syringe and dispense the blood into culture bottle in the vicinity of a flame.

(vi) Using an ethanol/ether swab, wipe the top of culture bottle and replace the cap. Gently mix the blood with the broth (Blood/broth volume should be atleast 1:10).

N.B.: The blood must not be allowed to clot in the culture media because any bacteria will become trapped in the clot.

(vii) Label the bottle with name, registration number of patient, ward, unit, date and time of collection.

(viii) As early as possible, incubate the inoculated media at 35 ° — 37° C.

 

INCUBATION

Incubate at 35°C — 37°C for upto 7 days with regular examining and subculturing as per guidelines.

 

SIGNS OF BACTERIAL GROWTH

Examine daily for upto 7 days.

(i) Turbidity above the red cell layer.

(ii) Haemolysis of the red blood cells.

(iii) Gas bubbles.

(iv)Appearance of small colonies in the broth on the surface of sedimented red cell layer or along the wall of bottle.

A sterile culture usually remains clear. A slight turbidity may develop after several days of incubation. 

lf there are signs of bacterial growth, subculture the broth and examine a Gram stained smear for bacteria and then do standard follow up for identification of organism.

 

BACTEC

• 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 BACTEC 9000 series of blood culture instruments are designed for the rapid detection of microorganisms in clinical specimens. The sample to be tested is inoculated into the vial which is entered into the BACTEC instrument for incubation and periodic reading.

• Each vial contains a sensor which responds to the concentration of CO2 produced by the metabolism of microorganisms or the consumption of oxygen needed for the growth of microorganisms. 

• The sensor is monitored by the instrument every ten minutes for an increase in its fluorescence, which is proportional to the increasing amount of CO2 or the decreasing amount of O2 present in the vial. 

• A positive reading indicates the presumptive presence of viable microorganisms in the vial.

 

COLLECTION AND TRANSPORT OF VARIOUS BODY FLUIDS

 

COLLECTION AND TRANSPORT OF EFFUSIONS

• Synovial, Pleural, Pericardial, Ascitic and Hydrocele fluids
• An effusion is fluid which collects in a body cavity. Fluids which collected due to an inflammatory process is referred as an exudate and that which forms due to a noninflammatory process is referred as a transudate.

• Collection and transport of effusions:
• After aspiration, aseptically dispense the fluid as follows:

• 2 — 3 ml into dry, sterile, screw — cap tube or bottle.

• 9 ml into a screw — cap tube or bottle which contains l ml of sodium citrate solution. Mix the fluid with the anti - coagulant.

• An anticoagulant is required to prevent clotting, especially exudates. The citrated samples can be used for the cell count, protein estimation, microscopy and culture. A sample without citrate is useful to see whether clotting occurs.

• Label it properly and deliver the specimen with a request form to the laboratory as early as possible.

• If there is delay in transport of specimen, then dispense 5 ml of fluid into a bottle of sterile thioglycollate broth and mix.

 

COLLECTION AND TRANSPORT OF CSF

• Cerebrospinal fluid must be collected by an experienced Medical Officer or health worker. It must be collected aseptically to prevent organisms being introduced into the central nervous system. The fluid is usually collected from the arachnoid space.

• A sterile wide bore needle is inserted between the fourth and fifth lumbar vertebrae and the CSF is allowed to drip into a dry sterile container. A ventricular puncture is sometimes performed to collect CSF from infants.

• CSF should be examined as early as possible. A delay in examining CSF reduces the chances of isolating a pathogen. It will also result in a lower cell count due to WBCs being lysed, and to a falsely low glucose value due to glycolysis.

• When trypanosomes are present, they will be difficult to find because they are rapidly lyzed once the CSF has been withdrawn.

 

COLLECTION OF CSF

1.    Take two sterile, dry, screw-capped containers and label one No.1 (first sample for culture purpose) and No.2 (second sample for other investigations).

2.    Collect about 1 ml of CSF in container No.1 and about 2-3 ml in container No.2.

3.    Immediately deliver the samples with a request form to the laboratory.

COMMON PATHOGENS IN CSF

BACTERIA	GRAM POSITIVE: Streptococcus pneumoniae, Streptococcus agalactiae, Listeria monocytogenes.
	GRAM NEGATIVE: Neisseria meningitides, Haemophilus influenza type b, Escherichia coli, Pseudomonas aeruginosa, Proteus species, Salmonella species,Flavobacterium meningosepticum
OTHER BACTERIA	Mycobacterium tuberculosis, Treponema pallidum
FUNGI	Cryptococcus neoformans (mainly in AIDS patients) and less commonly Aspergillus species
VIRUSES	Coxsackieviruses, echovirus, and arboviruses, herpes simplex 2 virus, varicella zoster virus and Lymphocytic choreomeningitis virus (LCM). Rarely polio viruses.
PARASITES	Trypanosoma species and Naegleria fowleri. Also Toxoplasma gondii (in AIDS patients)