This project report summarizes the process of blood culture testing. The purpose is to detect bloodborne microorganisms in patients with sepsis. Blood samples are inoculated into culture bottles and incubated for 5 days. The system monitors for increases in fluorescence that indicate growth. Positive bottles are subcultured onto agar plates and identified using staining techniques like Gram stain and Vitek 2 compact system. Identification of bacteria and reporting of antibiotic sensitivities helps in diagnosis and treatment of septic patients.

1. PROJECT REPORT OF
BLOOD CULTURE

2. BLOOD CULTURE

3. INTRODUCTION
Detection of microorganisms in a patient’s blood has
diagnostic and prognostic importance. Blood
are essential in the diagnosis and treatment of the
etiologic agents of sepsis. Bacterial sepsis constitutes
one of the most serious infectious diseases and,
therefore, the expeditious detection and
of blood borne bacterial pathogens is an important
function of the diagnostic microbiology laboratory

4. PURPOSE OF EXAMINATION
To detect presence of
microorganisms in bacteremic
and septicemic patients

5. SPECIMEN REQUIREMENTS ANS STORAGE OF
SPECIMEN
Blood / Body fluids from bacteremic and
septicemic patients, optimum volume being 8-
10mL in adults and 1-3 mL in paediatric patients.

6. STORAGE INSTRUCTIONS
Bactec blood culture bottles are stored at 2-
25 C in a dry location out of direct sunlight.
EQUIPMENTS AND MATERIALS
Bactec 9050
Vitek 2 Compact

7. PROCEDURE
•Label all vials. Do not write on or place any labels over the Bactec vial barcode, as this is
used by the instrument to process the specimen.
Inoculated vials should be placed into the BACTEC 9050 series
instrument as soon as possible for incubation and monitoring.
•Vials entered into the instruments will be automatically tested for the
duration of the testing protocol i.e. 5 days. The BACTEC Fluorescent
System will identify positive vials. The sensor inside the vial may not
appear visibly different in positive or negative vials, however, the
BACTEC Fluorescent System can determine in sensor fluorescence.
•Positive vials should be subcultured and an appropriate smear
prepared. All positive vials should be handled using regular safety
practices and containment facilities.

8. Processing an instrument-positive vial
•Remove the vial from the instrument and invert vial to mix contents.
•In biological safety cabinet, vent the vial to equilibrate vial pressure
with atmosphere.
•Remove aliquot from vial for stain preparations.
•Inspect smear and report preliminary results only after smear
evaluation.
If at the end of incubation, an instrument
negative vial appears to be positive (i.e., bulging
septum, or very darkened blood),
it should be sub cultured, stained and treated
as a presumptive positive, provided the stain
result is positive.

9. Sub culturing of vial
1. Sub culturing should be performed in a biological safety cabinet.
2. Prior to sub culturing, place the vial in upright position. After decontaminating the
rubber septum with 70% alcohol, insert a sterile needle into the bottle.
3. Remove the needle after any pressure is released and before sampling the vial for
subculture.
4. The insertion and withdrawal of the needle is done in a straight-line motion, avoiding
any side-to-side motions, which could permanently damage the septum.
5. Do not re-cap the needle. Discard needles in a puncture-resistant biohazard container
after cutting the needle.
6. Subculture on appropriate media and incubate at 37 C.
7. Proceed with identification and sensitivity testing with Vitek 2 Compact.

11. REPORTING OF RESULTS
An instrument positive vial may be confirmed by Gram stain. A positive result indicates
the presumptive presence of viable microorganisms in the vial.
If smear is positive, subculture on appropriate media and refer procedures for reporting
respective organisms.
If no microorganisms are present on the smears, subculture to solid media, re-enter the
vial into the instrument as an ongoing negative vial immediately and allow to complete
the test protocol.

12. Fig. Growth of(a) Alcaligenes facecalis , (c) Pseudomonas stutzer
&(d)stenotrophomonas maltophilia on blood agar (b)Pseudomonas
aeruginosa on macConkey agar

13. IDENTIFICATION OF ORGANISM BY USING VITEK 2 COMPACT
This Section describes the VITEK 2 automated microbiology system and its application in the
identification of microorganisms.

14. VITEK 2 Test Kit
Reagent Cards
Reagent card

16. Some antibiotics which are not present in VITEK CARDS are applied
manually.
For E. coli and Klebsiella spp. antibiotics applied are
Norfloxacin
Netilmycin
Cefixime
Ofloxacin
Fig 8:Impregnation of the Antibiotic discs.
Fig 9: Antibiotic Sensitivity Testing by Kirby-Bauer disk diffusion method.

17.  Some Staining methods use for the detection of
bacteria and component present in the slid
1)Gram staining:-
⦁ 2)AFB staining :- Acid-Fast Stain
•Interpretation of Acid-Fast
Stain
•Acid fast: Bright red to intensive purple (B), Red, straight or slightly
curved rods, occurring singly or in small groups, may appear beaded
•Non-acid fast: Blue color (A)

18. Examples of Acid-Fast Stain
Mycobacterium tuberculosis visualization using the Ziehl–Neelsen stain.
Source: Wikipedia
•Acid-fast: Mycobacterium tuberculosis, Mycobacterium smegmatis.
•Non-Mycobacterial bacteria: Nocardia
•Coccidian Parasites: Cryptosporidium

19. Fig 2: Gram Positive Bacteria
Fig1:GRAM NEGATIVE BACTERIA

20. Thank You