LAB 4

BLOOD CULTURE

INTRODUCTION

Blood culture can be defined as a microbiological culture of blood.It is the most widely used in
the detection of bacteremia and fungemia that are spreading through the bloodstream.This is
because the bloodstream is a sterile environment. It is an important way to identify the etiology
of bloodstream infections and sepsis.Bacteremia is known as the presence of bacteria in the
blood which may develop into septicemia. Usually,common organisms found in the blood are
part of the normal flora such as Gram positive cocci,gram negative stems, Pseudomonas and
so on.

To set up the blood culture experiment,a certain amount of blood is collected and then it is
added to the bottle of medium with a specific media for aerobic and anaerobic
organisms.Medium that used for anaerobes is thioglycollate broth while for aerobes is trypticase
soy broth.Then, the changes that occur in the culture after the blood sample is mixed with the
broth actually indicate signs of the type of organism that present in the blood.

Next for cultivation media ,there are three agar that are used to identify the organism which
are MacConkey agar, Blood agar and Sabouraud dextrose agar.Then,the colonies observed will
be further examined by performing gram staining. Furthermore,to confirm the identity of the
organisms that present,there are several biochemical tests that are conducted after the gram
staining.

OBJECTIVES

1. To set up blood cultures with various media.

2. To read blood cultures reactions .
3. To design a scheme to identify the infective agent.
MATERIALS

1 Anaerobic Jar with Gas-Pak

1 set Gram stain reagent
1 Sabouraud dextrose agar plate
1 Blood agar plate
1 MacConkey Agar plate
1 bottle 50ml Thioglycollate broth
1 bottle 50ml Trypticase soy broth
1 bottle 10ml blood sample
Various reagents and test medium
PROCEDURES
OBSERVATIONS

1.PSEUDOMONAS AERUGINOSA

BLOOD CULTURES APPEARANCE

-The broth become cloudy

-There has turbidity from microbial growth

COLONY MORPHOLOGY
A) MACCONKEY AGAR

-Non lactose fermenters

-form flats and smooth colonies
-Colorless
 B) BLOOD AGAR

-Beta -hemolytic
-large colonies(-metallic sheen, mucoid, rough, or pigmented (pyocyanin) )

C) SABOURAUD DEXTROSE AGAR

Do not grow on SDA

GRAM STAINING

-Gram negative bacteria

-Appear as reddish or pink rods

Presumptive identifications:

Pseudomonas Aeruginosa
BIOCHEMICAL TEST
LIPASE TEST

-Pseudomonas aeruginosa positive for lipase production

-Showed clear surround the growth (RIGHT)
2. KLEBSIELLA PNEUMONIAE

COLONY MORPHOLOGY

A) MACCONKEY AGAR

-Lactose fermenter

-pink coloured

-mucoid

B) BLOOD AGAR

-Non-hemolytic (gamma hemolytic)

 - greyish white

C) SABOURAUD DEXTROSE AGAR

Do not grow on SDA

GRAM STAINING

-Gram negative bacteria

-Rod shaped bacteria

Presumptive identifications:
Klebsiella Pneumoniae
BIOCHEMICAL TEST

CITRATE TEST

-Color change from green to intense blue along the slant

DISCUSSION

Pseudomonas aeruginosa and Klebsiella pneumoniae are the bacteria that infect the
blood.Both of the bacteria cause septicemia.In this experiment, for the blood culture
appearance, Pseudomonas aeruginosa shows different appearance at the broth which is the
broth become cloudy.So that showed that the turbidity is actually from the microbial growth.

Then,from the broth,the bacteria was taken from it and then streaked on the medium that has
been prepared, which is MacConkey Agar, Blood agar and Sabouraud Dextrose agar
plate.MacConkey agar is a medium that designed to differentiate and isolate enterics based on
their ability to ferment lactose.If the organism is ferment lactose and produced an acidic
environment, it will appear pink because of the neutral red turned to red.Next,if the organism is
non-fermenters,it produced colorless or normally coloured colonies.Based on the result in this
experiment, Pseudomonas aeruginosa formed flats and smooth colonies and it is colorless.So
the bacteria is non lactose fermenters.For Klebsiella pneumoniae , it produced pink color and
mucoid.So this identified that it is lactose fermenters.

Furthermore, blood agar is used in cultivating fastidious organisms and for identifying the
hemolytic capabilities of an organism.There are some bacteria that produced exoenzymes that
lyse the red blood cell and lower the hemoglobin.So this is called hemolysin.Bacteria can
produced many different types of hemolysin.Beta-hemolysin is red blood cell and the
hemoglobin will be break down completely.Beta hemolysis is showed if it leaves a clear zone
around the bacterial growth on the agar plate.Other than that γ-hemolysis or gamma hemolysis
is identified when the organism do not produce hemolysin and also do not break down the blood
cells so there is no clearing occur.Based on the result above, Pseudomonas aeruginosa is a
Beta hemolytic because the colonies are large with metallic sheen ,mucoid, rough or
pigmented(pyocyanin). Next,Klebsiella pneumoniae appear as mucous colonies which the color
is greyish white on blood agar.So Klebsiella pneumoniae is classified as non-hemolytic or
gamma hemolytic.Both of the bacteria do not growth on Sabouraud dextrose agar .

After that, a single colony from the agar was taken to observe the gram stain.For
Pseudomonas aeruginosa,it appear as reddish or pink rods when doing the gram
staining.So,Pseudomonas aeruginosa is a gram-negative bacteria.Next,for Klebsiella
pneumoniae is also gram-negative bacteria.It also appear in rod shaped bacteria.
Lastly,to make sure the type of organism that has appeared in the blood,there has been some
biochemical test that has been done to confirm the identity of the organism.
Lipase test is one of the biochemical test that used to make sure that the bacteria is
Pseudomonas aeruginosa.Tributyrin agar test is used to perform lipase test.Tributyrin agar is
known as a differentiation medium that test the ability of organisms to produce exoenzymes
called lipase that hydrolyzed tributyrin oil.If the organism produced lipase,a clear halo appear
surrounds the area.So this show that the organism has been grown.Based on the result,
Pseudomonas aeruginosa is positive for lipase production.
The second biochemical test that is used is citrate test.This test is to confirm the organism is
Klebsiella Pneumoniae .Citrate test is known to test the ability of an organism to utilize citrate as
a source of energy. For this test,if the organism showed a positive reaction,the color along the
slant changed from green to the intense blue.While for the negative reactions, the slant
remained green.In this experiment,the Klebsiella pneumoniae showed positive reaction in this
test.Therefore,based on overall experiment,it can be confirmed that the present bacteria in the
blood sample are Pseudomonas aeruginosa and Klebsiella pneumoniae.

CONCLUSION

In a nutshell,this experiment showed that there were several methods that can be used to detect
any microorganism such as bacteria,virus or fungi that are present in the blood culture.So
indirectly,the disease that present in the patient’s blood also can be detected using this
experiment.Hence,the bacteria that identified in this blood sample are Pseudomonas
aeruginosa and Klebsiella pneumoniae.
References

1. Welcome to Microbugz - Lipase Test. (n.d.). -.

https://www.austincc.edu/microbugz/lipase_test.php

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of-klebsiella-pneumoniae/

3. Aryal, S. (2019a, June 14). Citrate Utilization Test- Principle, Media, Procedure and Result.

Microbiology Info.Com. https://microbiologyinfo.com/citrate-utilization-test-principle-

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