HEALTH

Sunday, 5 June 2011

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

MICROBIOLOGY- STOOL CULTURE AND SENSITIVITY


STOOL CULTURE

PRINCIPLE:

The isolation and identification enteric pathogens (Salmonella/Shigella and determination of their

 Personnel

Student Technician
Laboratory Assistant
Rotating
Technician
Technician
Technologist
Specimen Sorting and numbering
Culture

Interpretation of Culture

     *

   
     *

        *
Identification Tests
     
     *

Sensitivity Tests

     *
      
Reporting





*Under supervision only

Safety    wear gloves
              Open bench

Reception – stool specimens are receive in the laboratory on a suitable wide-mouth non-sterile container (mono wax) or in the form of a rectal swab. Specimen should promptly be delivered to the laboratory for processing.

Equipment/media
Inoculating loops, Dexoxycholate citrate Agar (DCA) or Salmonella Shigella Agar (SS),MacConkey with crystal violet, Selenite F broth, Triple Sugar Iron (TSI)Agar. Urea Agar slope peptone water.

Macroscopic examination:
Note the presence of mucus and blood



CULTURE
Note:      (i)    technologist to see Biochemical reaction & sensitivity plate before signing
                       of report.
               (ii)    isolates are to be kept on slopes. (for storage)
DAY 1

1.   Inoculate a loopful of the stool specimen (if loose or watery) or a peanut size if
      formed) on to DCA/SS Agar and MacConkey Agar (with 1 crystal violet). Also
      inoculate Selenite F broth with about 2 – 3 times the size of sample used for solid
      media. Incubate the seeded media overnight aerobically at 35 -37oC

DAY 2

EXAMINATION AND IDENTIFICATION

Routine identification of suspected Salmonella and shigella isolates:

1.      UREASE TEST using a sterile straight wire pick an individual clourless smooth colony with or without black spot in the centre on the DCA/SS and inoculate a tube of Peptone water and Urea Agar slope.
2.      Incubate at 35-37oC up to 4 hours examining at hourly intervals for a pink colour throughout the urea agar slope.
3.      If urease test is negative at 4 hours incolate a tube of TSI/KIA with a sterile straight wire from the peptone water tube or similar smooth colony stabbing first the butt and then streaking the slope. Close the tube with a loose-fitting cap and incubate at 35-37oC overnight.
4.      PURITY PLATE
inculate Macconkey plate with same colony as purity plate for serology.
5.      ENRICHMENT BROTH
Sub-culture the Selenite F broth culture on a plate of DCA/SS Agar and follow up as above on Day 3. this is necessary if no colonies resembling Salmonella spp are seen on the primary culture plates. Discard selenite F. broth.
       


DAY 3

1.    Examine the TSI/KIA Urea Agar slope, iodole etc for reactions   
       suggestive of Salmonella or Shigella.
2.   Test serologically and isolate giving reactions suggestive ofSalmonella 
       technique. Subculture on MacConkey for purity to make sure you are  
       dealing with non-lactose fermented.
3.    Perform sensitivity test on the isolate using appropriate antibiotic
       discs. Include a control E. coli strain on a separate sensitivity test agar
                                plate.

ADDITIONAL TESTS

For stool specimens pf infants up to 2 years inoculate one of the Lactose fermenting rose-pink colonies on MacCoonkey plate into Peptone water, kosers citrate, Urea Agar slope and TSI for the isolation of E. coli. If E. coli is isolated test serologically for the appropriate E. coli stains (ETEC,EIEC, EPEC or EHEC)

Summary of the laboratory examination of Raecal Specimens

DAY 1         Culture

Primary Plating Media          SS Agar or DCA 
                                               MacConkey with Crystal violet (children less than 2 years)

Enrichment:  Selenite F

DAY 2

1      Examine SS/DCA culture for NLF’s
        Salomnella species
        Shigella species

2.     Inoculate Peptone water & Urea
        If no Urease production after 4 hrs
        Inoculate      1.  TSI/KIA incubate at 35oC – 37oC.
              2.  MacConkey Agar for Purity and serology.


3.    Also subculture the selenite F culture on SS/DCA. This is important if there is not
        growth suggestive of Salmonella or shigella on the Primary Culture.
  
Cholera
If suspected, cholera specimens should be sent to the Public Health Reference Laboratory

Reporting
1. Report the type of Salmonella or Shigella spp isolated with the sensitivity on Day 4

2. If there was no suggestive isolate report as:
     No Salmonella or Shigella isolated.
BY BMS DZAMESI

MICROBIOLOGY- URINE CULTURE AND SENSITIVITY


URINE CULTURE AND SENSITIVITY

PRINCIPLE:

The isolation and identification of Significant bacteriuria in urine.  Sensitivity testing should be performed on the organism(s)

Personnel
Student Technician
Assistant Technician
Rotation Technician
Technician
Technologist
Specimen Sorting and numbering
*
Microscopy
*
Culture
*□
*
Interpretation of cultures


Sensitivity test


Reporting


*□
*Under supervision only.

Safety:  Wear gloves

Reception:  Inoculate immediately or refrigerated at 4ºC for not more than 18 hours after collection.

Equipment/Reagent:
Calibrated Standard loop (1/500) or 1/400).
Microscope Slides, Cover glass, Pasteur pipette, Beakers, Racks, Saline, CLED(or BA and Mac) DSTA, (CA) when needed, Peptone water Urea, Citrate, TSI, Antibiotic discs, Plasma, Gram/Z-N Stains when needed, test tubes, Sterile centrifuge tubes and Centrifuge.

DAY 1

Test Procedure:
(Semi-quantitative Standard loop Method)

Mid-Stream Urine (MSU)  is the usual specimen (see others below)
(1)     mix the urine sample carefully by rotation but not by inversion (to avoid any contamination if the lid is loose).
(2)     Dip the standard loop (1/400 or 1/500ml) vertically into the urine and inoculate onto ¼ Cystine Lactose, Electrolyte Deficient (CLED) Agar or Blood Agar (BA) and MacConkey Agar (Mac) plates.  Only the ring of the loop should enter the urine.
(3)     Incubate aerobically at 35-75ºC overnight (For N gonorrhoeae put deposit on Chocolate agar, (send deposit for TB culture to PHL)




                                                                                    Fig 1

Microscopy:
Put 2 drops of well mixed uncentrifuged urine onto a Slide.  Apply a cover slip.  Examine with a x 10, objective for WBC/RBC/Epithelial Cells and bacteria.  To identify type of cells or casts use x 40 objective.  Count number of cells in 10 fields and express the results as per High power field.

In emergency cases
  • Put up direct Sensitivity test on urine containing.
(1)    20 or more WBC per field
(2)    Bacteria
(3)    Both bacteria and 20 or more WBC

DAY 2
(1)    Examine plate.  Note the degree of growth on each ¼ plate (Fig 1)
(2)    Interpretation of culture
i.             No bacterial growth
ii.           No significant growth less than (<) 200 cfu
iii.         Significant growth:-
                  With a 1/400 ml loop approximately 250 cfu
                  With a 1/500 ml loop approximately 200 cfu
This is proportional to > 108 bacteria./Litre (.> 105 bacteria/ml)

(3)    Other considerations
a.       S. typhi – even a few colonies are significant
b.      Suprapubic aspirate) less than 105/ml is significant            Ureteric samples     )
c.       Candida spp – important in diabetics and immunosuppressed patients
d.      *Pseudomonas species….

(4)    Mixed growth
a.       Significant growth of only two types of colonies identify and do sensitivity test.
b.      More than two types of colonies, ask for a repeat specimen




Identification/Sensitivity Tests
Some Common Pathogens

Organism
Gram Result
Test(s)
*  * Sensitivity
E. coli, Klebsiella
Enterobacter
Citrobacter & Other
Coliforms
GNR (LFs)
Indole, Urea Citrate, TSI
Motility Tests or consider
direct API 20E
Ampicillin, Tetracycline Nalidixic Acid,
Nitrofurantoin Pipedidic Acid, Septrin
Gentamicin, Cefuroxime, Augmentin
Pseudomonas spp
GNR (NLFS0
Oxidase, motility
Non-fermenter of Sugars, or
Consider direct API 20E
Gentamicin, Noroxin, Cefotaxime,
Ofloxacin Ceftazidime, Amikacin
Urotractin, ciprofloxacin
Salmonella spp
GNR
(NLFs)
Indole, Urea Citrate TSI,
Motality or Consider direct
Using specific antisera
Chloramphenicol, Cefuroxime
Angmentin, Nalidixic Acid, Noroxin,
Urotractin Gentamicin, Cefuroxime
Nitrofurantoin, Ampicilin
Faecal Streptococcus
(enterococci)
GPC (Chains)
Catalase test, Growth on
MacConkey
As above

**  Alternatively, use available multodisc for Gram negatives or Gram positives

Reporting
                    I. Significant growth
Must be reported with sensitivities as follows

1.      Out patients  - Report oral antibiotics only and keep the Sensitivity of parenteral ones on the request forms.
2.      In-patients – Report both parenteral and oral antibiotics.
List of antibiotics will be amended from time to time, depending on availability.

II.                No significant growth.

III.             Mixed growth
a.       Significant growth of 2 types of organisms – report with sensitivity
b.      More than 2 types of organisms, report as mixed growth.

Repeat culture requested
BY BMS DZAMESI