This document discusses procedures for analyzing bacterial colonies from blood samples and fungal samples from sputum. For blood samples, it describes how blood cultures are used to identify bacteria or fungi in the bloodstream. It also discusses antibiotic sensitivity testing and serological tests. For sputum samples, it discusses potassium hydroxide testing and culturing samples on Sabouraud dextrose agar to identify infectious fungi. Identification may involve gram staining, examining hyphae and spores under a microscope.

2. Content
• Introduction
• ANALYSIS OF BACTERIAL COLONY IN BLOOD
SAMPLE
- Blood Culture
- Antibiotic Sensitivity of bacteria:
- Serological tests
• FUNGAL ANALYSIS OF SPUTUM SAMPLE
• Result
• Conclusion

3. INTRODUCTION
 ANALYSIS OF BACTERIAL COLONY IN BLOOD
SAMPLE:
• BLOOD CULTURE :
A blood culture is a microbiological culture of blood. It is a sensitivity test that
helps to identify the foreign invader like bacteria, yeast and microorganisms in
blood. It is vital for identifying pathogen causing serious infection. Having these
pathogen in blood stream can be a sign of a blood infection. A condition known
as bacteremia.
BACTEREMIA:- Bacteremia is an infection, caused by bacteria, that enters the
bloodstream.
It is the most important way to diagnose the etiology of bloodstream infections
.

4. Principle:-
A blood culture is a laboratory test in which blood, taken from the patient,
and inoculated into bottles containing culture media (BHI supplement) to
determine whether infection-causing microorganisms (bacteria or fungi) are
present in the patient’s bloodstream.
BHI:- Brain heart infusion (BHI) is a growth medium for growing
microorganisms. It is a nutrient-rich medium, and can therefore be used to
culture a variety of fastidious organisms.
BHI bottle are of 70ml for adult and 30ml for child in which 1-2ml of patients
blood are added to the culture media (BHI) for inoculation.

5. Procedure:-
Inoculate 1-2 ml blood sample, in culture media (BHI supplement)
After 2-7 Days
Media is streaked on Chocolate/ blood agar and on macconkey agar
Incubate for 24 Hrs.
After 24 hrs. the growth of bacteria is seen on the both agar plate
Make a Smear
Gram’s staining of bacteria
Observe the smear and preform biochemical test eg.
Catalase test
Citrate test
Urease test
Phenyl Pyruvic Acid (PPA)
Methyl red (MR)

6. o CHOCOLATE AGAR
• Growth of bacteria colonies are observed on Chocolate agar.
o MACCONKEY AGAR
• Whitish colonies of bacteria are observed which indicates the presence of
Gram Negative Bacteria on the Macconkey Agar plate
OBSERVATION:

7. TESTS
Catalase
Test
Citrate
Test
Urease
Test
Phenyl
Pyruvic Acid
OBSERVATION -
(Negative result)
+
(Positive result)
+
(Positive result)
+
(Positive result)
o BIOCHEMICAL TEST FOR GRAM NEGATIVE BACTERIA:

8. For sensitivity of bacteria
small amount of bacterial Colony are picked up by wooden swab.
Insert the swab in the test tube containing peptone and mix it gently.
Now by swabbing apply the bacterial Colony on MHA plate.
Small Wafers containing antibiotics are place on a MHA plate
After 24 Hrs.
bacteria are sensitive to the antibiotic, a clean ring, or zone of inhibition,
is seen around the wafer
Antibiotic Sensitivity of bacteria:
In antibiotic sensitivity, Antibiotic have been placed on an Mueller Hinton
Agar plate growing bacteria. Bacteria are not able to grow around
antibiotics to which they are sensitive and if bacteria is
grown around the antibiotic then we conclude that the
bacteria have resistance towards the particular antibiotic.

9.  Serologic tests
Serological tests are blood tests used for identification of antibodies
in blood sample.
1. Widal Slide Test:
Widal test is a serological method to diagnose enteric fever or typhoid which
is caused by the infection with pathogenic microorganisms like
Salmonella typhi, Salmonella paratyphi A, B and C.
It was developed by F Widal in 1896. Antibodies to Salmonella may be
detected in the patient serum from the second week after onset of infection.
When the Salmonella antigen are mixed/ incubated with
patient serum, anti- Salmonella antibodies of patient
serum react with the antigen and form agglutination.

10. Procedure :
Label the circles in the test card as O, H, AH, BH, Negative control and Positive control
Place One drop of positive control and negative control serum on the slide.
Now place a drop of antigen O,H, AH, BH on its reaction circle of the same slide
Place One drop of patients serum to each of the reaction circle
Mix contents of each circle uniformly over the entire circle with separate mixing stick.
Rock the slide gently back and forth observe the agglutination after 1 min.

11. 3. HBsAg :
Virucheck is the one step test for HBsAg. It is a rapid, qualitative, two side
sandwich immunoassay for the detection of hepatitis B surface antigen.
Blood containing hepatitis B virus (HBV) is a potentially infectious. Hepatitis B
surface antigen (HBsAg),, is the first serological markers that circulate in the blood
of infected person even 2 to 3 weeks prior to the appearance of clinical symptom.
Procedure :
• Dispense to drop of serum / plasma sample into the sample well ‘S' using the
applicator provided.
• At the end of 15 minutes read the result.

12. • NEGATIVE : A coloured band appear only at the control region ‘C'.
• POSITIVE : In addition to the control band, a coloured band also appear at
the test region ‘T'.
• INVALID : The test should be considered invalid if no coloured band appear
on the device. The test should also be considered invalid if the
colour band appear only at the test region
and not at the control region.

13. 4. Hepatitis C virus (HCV) :
HCV TRI-DOT is a rapid, visual, sensitive and qualitative in vitro Diagnostic test
for the detection of antibodies to Hepatitis C virus in human serum or plasma.
HCV TRI-DOT has been developed and designed with increased sensitivity by
using a unique combination of modified HCV antigens.
The device (an immuno - filtration membrane) include two test dots “T1” and “T2”
and a Built in Quality Control Dot “C”. The control dot will always develop colour
during the test, thereby confirming proper functioning of the device.
Procedure:
• While adding sample to the device, allow each solution to soak in before adding the next
solution.
• Add Three drops of buffer solution to the Centre of the device.
• Add one drop of patients sample using the sample drop provided.
• Add 5 drop of buffer solution.
• Add 2 drop of protein-A conjugate.
• Again add 5 drop of buffer solution.
• Test dots “T1” and “T2” either dark or Light in colour (pink) should be considered reactive for
antibodies to HCV.

14. At the end, the result is as follows:
NON REACTIVE RESULT:
Appearance of only one dot at the control region “C” indicates
that the sample is non reactive for antibodies to HCV.
REACTIVE RESULT:
• Appearance of 2 dot, one at the control region “C” and other at the test region “T1”
indicates that the sample is reactive for antibodies to HCV.
• Appearance of 2 dots, one at the control region “C” and other at the test region
“T2” indicates that the sample is reactive for antibodies to HCV.
• Appearance of all the three dots, one each at “C” T1” and “T2” region indicate that
the sample is reactive for antibodies to HCV.
INVALID RESULT:
If no dot appears after the completion of test, either with clear background or with
complete pink / purpose background the test indicate error.

15.  FUNGAL ANALYSIS OF SPUTUM SAMPLE
• Fungi are eukaryotic, heterotrophic, non photosynthetic organisms.
• The majority consists of microscopic filaments called hyphae, and the
network of filaments is the mycelium.
• Yeast form pasty and creamy colony under 37°(Optimum temperature)
where Mold form cottony and feathery colonies under 22°-25°
(Optimum temperature).
• Some fungus are dimorphic so they are culture under both
temperature

16. IDENTIFICATION OF FUNGAL INFECTION IN SPUTUM SAMPLE
• KOH Test:
• Potassium hydroxide (KOH) preparation is used for the rapid detection of fungal
elements in clinical specimen.
• KOH is a strong alkali. When specimen such as skin, hair, nails or sputum is mixed
with KOH , the clear visible hyphae and conidia of fungi are seen under microscope.
• KOH is mixed in equal proportion with the specimen on a sterile slide.
• Then the specimen material is teased with sterile inoculating needle, a coverslip is
placed over it and heat gently to remove air bubble,
• allow to fix for few minutes according to given specimen,
.

17. USES OF DIFFERENT CONCENTRATION OF KOH :
10%KOH 20%KOH 40%KOH
SPECIMEN TIME SPECIMEN TIME SPECIMEN TIME
Skin scraping 5 - 10 min Soft tissue 10 - 15 min Nail scraping 10 – 60 min
Sputum Sample 5 - 7 min Tissue biopsy 10 – 20 min
All fluids 5 min
Pus sample 5 - 7 min
Hair 5 - 10 min
Sputum Sample are culture for identification of infectious fungus.
• Under the Microscope Long branch - Like tubular structures
called HYPHAE are observed.
• Show the presence of spores formed directly from the hyphae.
• Hence Dermatophytes or Yeast are seen on a KOH test
indicates the person has a fungal infection.

18. • Sabouraud dextrose Agar :
Sabouraud Dextrose Agar (SDA) is a selective medium primarily used for the
isolation of dermatophytes, other fungi and yeasts.
The acidic pH of this medium inhibit the growth of bacteria but permits the growth
of yeasts and most filamentous fungi.
Procedure
• Streak the specimen on the medium with a sterile inoculating loop to isolated colonies.
• Incubate the plates at 22 – 37°C in an inverted position (agar side up) with increased humidity.
• Cultures should be examined at least weekly for fungal growth and should be held for 4 -6
weeks.
• After the growth of fungus on culture media, Gram staining are done
(Gram positive Fungus =yeast and Gram Negative Fungus = mold)
OBSERVATION:
• SDA is a agar media used to culture the fungi or yeast.
• Initially white spore form then turn into black dark
pigmented conidia in large number.

19. • Lactophenol Cotton Blue(LPCB) :
The Lactophenol Cotton Blue (LPCB) wet mount is most widely used method
for the staining and observation of fungi.
LPCB is a stain used for making semi-permanent microscopic preparation of fungi
The LPCB stain has following three components :
• Phenol : Kills any live organism.
• Lactic acid : Preserves fungal structures.
• Cotton blue : Stains the chitin and cellulose of the fungal cell wall intensely blue.
PROCEDURE :
• Place a drop of 70% ethanol on a clean microscopic glass slide
• Immerse the specimen in the drop of ethanol
• Add one or two drops of the LPCB before the alcohol dries out
• Place the coverslip gently avoiding air bubbles
• This preparation is now ready for examination
• Make the initial examination using low power objective. Switch to higher power (40X)
objective for more detailed examination of spores and other structures
Hyphae and spores are stained by LPCB
Young growth of fungus

20. Result :
o BLOOD CULTURE :
• Gram Negative bacteria are observed by Macconkey agar.
• From biochemical test Proteus mirabilis (urease test) are observe
• Proteus mirabilis. are sensitive for. Penicillin (Ticarcillin), Ciprofloxacin.
o Serological test:
• Blood sample A
 Widal slide test:
• Observe Agglutination or formation of clumps in the well. .
• Test well shows agglutination with H and AH antigen.
• Hence the infecting organism is Salmonella paratyphi A
• The patient is diagnosed with typhoid fever.

21. • Blood sample B
 HBsAg :
• Two distinct coloured line appear. i.e. Control region (C) and Test region (T).
• Indicates the presence of hepatitis B surface antigen.
• Blood sample C
Hepatitis C virus (HCV) :
• Appearance of only one dot at the control region “C”.
• indicates that the sample is non reactive for antibodies to HCV.
o FUNGAL ANALYSIS OF SPUTUM SAMPLE
• By KOH Preparation of SPUTUM presence of fungal infection are observed.
• From SDA and LPCB mount, Aspergills niger are seen.

22. Conclusion:
This is to conclude that