MICROBIOLOGY- BLOOD CULTURE


BLOOD CULTURE

PRINCIPLE:

            The isolation and identification of all organisms which may cause infection
            The determination of their sensitivities.

Personnel
Student Technician
Laboratory Assistant
Rotating Technician
Technician
Technologist
Specimen sorting and numbering
Subculture
Interpretation of Culture
*
*
Identification
*
*
Sensitivity
*
*
Reporting


*
*


*Under supervision only

Safety                          Wear gloves
                        -           Aseptic technique/Class II safety cabinet

Reception        -           Incubate blood bottles immediately at 37ºC

Equipment/reagents-   Glass slides, Media, anaerobic jar, Gaspak and Gram stain.

DAY OF RECEIPT

            a.         Note date and number of specimen
            b.         Incubate immediately at 35ºC.

DAY ONE (after 24hrs incubation)

                                    Inspect all bottles for evidence of growth

NOTE:                        -    Uniform or subsurface turbidity
-          evidence of haemolysis
-          presence of colonies in the blood layer
-          gas production
-          coagulation of the bottle
-          a surface pellicle
-          etc.

a.         Gram staining

            The bottle is opened aseptically, a small amount of broth is removed with a sterile loop or Pasteur pipette, and a Gram stained smear is examined for the presence of microorganisms and subcultured on  an appropriate media.

b.         Subculture
           
            All bottles onto BAo2, BAano2, MacConkey for 18-24hrs
            Add CA + 5% Co2 for 48hrs if N. meningitidis is suspected or Gram stain show   Gram negative diplococci.
            Add Sabourand in suspected candida infection.

c.         Direct-sensitivity
           
            Using blood culture medium (positive growth) as inocular if necessary.

d.         Re-incubate all bottles

DAY TWO:   a.         Signed preliminary reports to wards (CLSCP)
                       
(i)                 NBG after 48hrs incubation for all Negatives
(ii)               Gram report for positives stating further report to follow

b.         Indicate on the request card that the Preliminary Report has been                sent.

     c.          Do further identification tests on Positives plus sensitivity test.                               The choice of antibiotic disks will depend on the result of the                                  Gram stain.



DAY THREE     a.    Read biochemical results an write reports
b.     Record all signed reports in the book and send report to the ward
(CLSCP) Central Laboratory &Specimen Collection Point.


NOTE:                       1.         Examine all bottles daily for evidence of growth, and do                                                                 subculture after 7 days incubation.
                       
                                    2.         Discard positive plates after results have been signed

BACTERIAL ENDOCARDITIS/VALVE ABNORMALITY

These culture bottles are incubated for a total of 3 weeks, where (3) cultures have been received.  Bottles should be subcultured on days 2, 7, 14, and 21.

BRUCELLOSIS

            Bottles should be incubated for a total of six(6) weeks.
            All manipulation must be performed in the microbiological safety cabinet.
            Biochemical Tests – Indole, Urea, Citrate, TSI, Motility Test.

GRAM RESULT
PLATES AND TESTS
DIRECT SENSITIVITY
GPC?
Staphylococcus
BA1-02, CACO2 Ano2
Sensitivity + Methicillin at 30ºC
Direct tube coagulase
*Penicillin, Tetracycline,
Erythromycin, Gentamicin,
Cefuroxime, Methicillin
GPC? Streptococcus
BA1-O2, CA Co2 Ano2
Test, Sensitivity on BAO2
Direct Strept. Grouping
**Latex for Strept. Pneumonia
*Penicillin, Ampicillin
Erythromycin, Tetracycline
GNR – Large
?Coliform
?Pseudomonas
 BA1-O2, CA Co2 Ano2
MacConkey
NLF check oxidase
If negative oxidase, other Gneg.tests
Check Pso/PSH/Vi
*Ampicillin, Cefuroxime,
Ciprofloxacin
Gentamicin
Ceftazidime
Gentamicin Chloramphenicol
GNR – small?
HI
CA CO2, BA Ano2
Direct HI latex agglutination
Requirement for X & V

  • For patients on antibiotic treatment, set up sensitivity tests for those drugs
when available




DIRECT LATEX FOR HI(Hinfluenae) 1 drop supernatant + 1 drop of latex
DIRECT TUBE COAGULASE        -           1 drop of blood/broth into diluted plasma
DIRECT STREPT. GROUPING       -           1 drop of blood/broth against each group.
BIOCHEMICAL TEST                     Indole, Urea, Citrate, TSI + motility Test


TO SUBCULTURE BOTTLES

1.      Gently shake bottles to resuspend the erythrocytes
2.      Using a loop, subculture onto BA02, BAAno2CA + 5% and MacConkey.  Incubate for 24-48hrs.


Final subculture

Do final subculture and discard bottles after 7 days incubation except for suspected brucellosis and endocarditis(see below)

Note:  i.           Technologist to see Biochemical reaction & Sensitivity plate before                                     signing of report

            Ii          Isolates are to be kept on slopes.(for storage)


Reporting:

  1. Preliminary report
  2. Final report
a.              Significant growth with Sensitivities
b.              Mixed growth (Indicate types of organisms in day book)
    
       Request repeat specimen DISCUSS WITH TECHNOLOGIST
BY BMS DZAMESI

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