ARSI UNIVERSITY COLLEGE OF HEALTH SCIENCES

DEPARTEMENT OF MED LABORATORY

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 Group Members
1. Marifa Abdela
2. Mekides Hailu

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 Topic

Discuss on bacteriological techniques (Gram stain

& Culture) commonly used in diagnosis of
bacterial infection.

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 Introduction

Medical Bacteriology: - it involves the study of

pathogens, the disease caused by them, and the
body’s defenses against disease.

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 bacteriological techniques in Culture
the different bacteriological techniques in culture
 gram stain
 culturing bacterial
 biochemical tests
 antimicrobial agents & susceptibility testing

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 Gram’s stain

Introduction
This method was developed by the Danish bacteriologist
Christian Gram in 1984.
 Most bacteria are differentiated by their gram reaction due to

differences in their cell wall structure.

 The surface of bacterial cell has got a negative charge due to the

presence of polysaccharides and lipids (PG) this has made the

surface of the bacteria to have affinity to cationic or basic dyes
(when the colouring part is contained in the basic part.)
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 Principle

The principle of Gram’s stain is that cells are first fixed to

slide by heat or alcohol and stained with a basic dye (e.g. crystal
violate), which is taken up in similar amounts by all bacteria. The
slides are then treated with an Gram’s iodine (iodine KI
mixture) to fix (mordant) the crystal violet stain on Gram positive
bacteria, decolorized with acetone or alcohol, and finally counter
stained with Safranin.

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 Required reagents

Crystal violet

Gram’s Iodine

Acetone-Alcohol

Safranin

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 Procedure

1. Prepare smear from specimen or culture.

2. Allow the smear to air-dry.

3. Rapidly pass slide three times through flame.

4. Cover fixed smear with crystal violet for one minute

and wash with tap water.

5. Tip off the water and cover the smear with Gram’s
iodine for one minute.
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 Cont….
[Link] off iodine solution with tap water.

7. Decolorize with acetone-alcohol for 30 seconds.

8. Wash off the acetone-alcohol with clean water.

9. Cover the smear with safranin for one minute.

10. Wash off the stain and wipe the back of the slide. Let the

smear air-dry.

11. Examine the stained smear with oil immersion objective to

look for bacteria.
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 Cont…
Results
 Gram positive bacteria …………………….. purple

Yeast cells …………………………………. Dark purple

Gram negative bacteria …………………….. Pale to red

 Nuclei of pus cell …………………………… Red

Epithelia cells ………………………………. Pale red

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 Cont….
Reporting of Gram’s stained smear

The report should include the following:

1. Gram reaction of the bacteria whether Gram

positive or Gram negative
2. Morphology and arrangement of the bacteria

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 Culturing bacterial Pathogen

Introduction

The purpose of using cultural techniques in

microbiology is to demonstrate the presence of
organisms which may be causing disease, and when
indicated, to test the susceptibility of pathogens to
antimicrobial agents.

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 DIFFERENT TYPES OF CULTURE MEDIA
For a culture medium to be successful in growing the
pathogen sought it must provide all essential nutrients,
ions, and moisture, maintain the correct pH and osmotic
pressure, and neutralize any toxic materials produced.
The main types of culture media are:
 Basic/simple
 Enriched
 Selective
 Indicator
 Transport
 Identification

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 Cont….
Basic media: These are simple media such as nutrient
agar and nutrient broth that will support the growth of
microorganisms that do not have special nutritional
requirements.
Enriched media: Enriched media are required for the
growth of organisms with exacting growth
requirements such as H. influenzae, Neisseria species,
and some Streptococcus species.

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 Cont…
Enrichment media: This term is usually applied to
fluid selective media which contain substances that
inhibit the growth of unwanted organisms,

Selective media: These are solid media which contain

substances (e.g. bile salts or other chemicals, dyes,
antibiotics)
which inhibit the growth of one organism to allow the
growth of another to be more clearly demonstrated. as
TCBS agar.

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 Cont….
Indicator (differential) media: These are media to
which dyes or other substances are added to
differentiate microorganisms.
e.g. MacConkey agar.
Note: Many media used to isolate pathogens are both
selective and enrichment or both selective and
differential.

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 Cont…
 Transport media: These are mostly semisolid media that
contain ingredients to prevent the overgrowth of
commensalism and ensure the survival of aerobic and anaerobic
pathogens
 Cary-Blair medium for preserving enteric pathogens and
 Amies transport medium for ensuring the viability of
gonococci.
 Identification media: These include media to which substrates
or chemicals are added to help identify bacteria isolated on
primary cultures.
 Examples include peptone water sugars,
urea broth, and Kligler iron agar..

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 Biochemical tests
Introduction
Biochemical tests are used to differentiate different

organisms based on their genus and species

characteristics.
Biochemical tests are performed on pure culture.

The following are some of the common biochemical

tests used for differentiation of different bacteria.

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 Catalase test
This test is used to differentiate those bacteria that produce
the enzyme catalase such as staphylococci from non
Catalase producing bacteria such as streptococci.
Principle
Catalase acts as a catalyst in the breakdown of hydrogen
peroxide to oxygen and water.
An organism is tested for catalase production by
bringing it in to contact with hydrogen peroxide.
Bubbles of oxygen are released if the organism is a
catalase producer. The culture should not be more than
24 hour old.

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 Cont…
Results
 Active bubbling ----------------- Positive test
Catalase produced
 No release of bubbles ---------- Negative test
No catalase produced
Control
Positive catalase control – staphylococcus species
Negative catalase control – streptococcus species

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 Coagulase Test
This test is used to differentiate Staphylococcus aureus
which produces the enzyme coagulase, from
[Link] and [Link] which do not
produce coagulase.
Principle
Coagulase causes plasma to clot by converting
fibrinogen
Results
to fibrin
Positive coagulase control ……………..Staphylococcus
aureus
Negative coagulase control ……… [Link] or Staph
epidermides

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 Urease test
This test is used to detect the enzyme urease, which
breaks down urea into ammonia.
Testing for urease enzyme activity is important in
differentiating enterobacteria. Proteus strains are
strong urease producers.
Principle
The test organism is cultured in a medium which
contains urea and the indicator phenol red.
When the strain is urease-producing, the enzyme
will break down the urea (by hydrolysis) to give
ammonia and carbon dioxide.

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 Cont…
 With the release of ammonia, the medium becomes alkaline
as shown by a change in color of the indicator to pink-red
Results in color
 Red/purple color…………….positive urease test
 Yellow/orange……………….. Negative urease test
Control
Positive urease control..............Proteus spp,
Negative urease control…………Salmonellae

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 Cont…

Fig. Urease test: The tube on the left is a

positive reaction; the tube in the middle is a
negative reaction and the tube on the right is
25 an un-inoculated control.
 Iodole test
 Testing for iodole production is important in the
identification of enterobacteria.
 Most strains of E. coli, P. vulgaris, P. rettgeri, M. morganii,
and Providencia species are indole positive organisms
Principle
The test organism is cultured in a medium which
contains tryptophan.
Iodole production is detected by Kovac’s or Ehrlich’s
reagent which contains 4(p)-dimethylamino-
benzaldehyde.
This reacts with the iodole to produce a red colored
compound.
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 Cont…
Results
 Red surface layer…………………Positive indole test
 No red surface layer………………Negative indole test

Control
Positive control ….. Escherichia coli
Negative control…. Klebsiella pneumoniae

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 Cont…

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 Citrate utilization test
The test detect the ability of an organism to use citrate as
its only source of carbon. This test is one of several
techniques used occasionally to assist in the
identification of enteric bacteria
Results
 Bright blue--------------------Positive citrate test
 No change in color of media--------Negative citrate test
Controls
Positive control --------------------- klebsiella
pneumoniae
Negative control---------------------Escherichia coli

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 Cont

Fig. Citrate utilization test: Left tube is a

negative result. Right tube is a positive result
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 Triple sugar Iron (TSI)
Looks at fermentation of glucose, lactose, and sucrose
and checks if hydrogen sulfide and gas is produced in
the process.
Basically a pH indicator will change the color of the
media in response to fermentation.
Result
Slant color red …………. does not ferment either
lactose or sucrose
Slant color yellow………Ferments lactose and/or
sucrose
Butt color red………….no fermentation of glucose

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 Cont…
Butt colour yellow…………. some fermentation of
glucose has occurred and acid has been produced
Cracks seen in the agar, ……………Gas formed
bubbles occured, or the entire slant pushed out of the
tube.
Blackening in the Butt…. H2S has been produced

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 Cont…

A B C D E F G

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 Cont…
From left to right:
A. Uninoculated control
B. Red slant and red butt, no black color= no fermentation
of glucose, sucrose or lactose. No Hydrogen sulfide
produced
C. Red slant and black butt= no lactose or sucrose
fermentation, H2S has been produced
D. Red slant with yellow butt= no lactose or sucrose
fermentation, Glucose is fermented, no H2S has been
produced
E. Yellow slant, yellow butt and black coloration= Lactose,
sucrose and glucose fermented, and H2S has been produced
F. Yellow slant, yellow butt and lifting and/or cracking of
media, no black coloration= Lactose, sucrose and glucose
fermented, H2S has not been produced but gas has been
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produced
G. Yellow slant, yellow butt and no lifting and/or cracking
 Antimicrobial agent
Introduction

Antimicrobial agent: A general term for drugs,

chemicals, or other substances that either kill or slow
the growth of microbes. Among the antimicrobial
agents are antibacterial drugs, antiviral agents,
antifungal agents, and antiparasitic drugs.

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 ANTIMICROBIAL SENSITIVITY
(SUSCEPTIBILITY) TESTING
The test is used to measure the ability of the drug
to inhibit or kill pathogens in vitro. I.e. it is used to
select effective antimicrobial drugs.
Sensitivity test is performed:
 For organisms with variable antibiotic sensitivity
(un predictable sensitivity) E.g. Shigella
 For non responding patients after taking adequate
therapy.
 For patients whose immune system is depressed
 For relapsing cases (reappearance of disease)

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 Disk Diffusion Sensitivity Testing

Principle: -
A disk of blotting paper is impregnated with a known
volume and appropriate concentration of an antimicrobial, and
this is placed on a plate of sensitivity testing agar uniformly
inoculated with the test organism. The antimicrobial diffuses
from the disc into the medium and the growth of the test
organism is inhibited at a distance from a disc that is related to
the sensitivity of the organism.

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 Cont..
The zone of inhibition is measured by clippers or ruler
and values are matched (compared) with the
predetermined standard values and reported as
susceptible, Intermediate and resistant.

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 Mueller Hinton Sensitivity testing Agar

Prepare the medium as instructed by the

manufacturer.
The PH of the medium should be 7.2 – 7.4.
Pour into 90mm diameter sterile Petri dishes to a
depth of 4mm. (i.e. about 25ml of Mueller Hinton
agar per plate]
Care must be taken to pour the plates on a level
surface so that the depth of the medium is uniform.

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 Procedure
1. Using a sterile wire loop, touch 3 – 5 well – isolated
colonies of similar appearance to the test organism and
emulsify in 3 – 4 ml to sterile physiological saline or
nutrient broth.
2. In a good light match the turbidity of the suspension to
the turbidity standard (mix the standard before use)
3. Using a sterile swab, inoculate a plate of Muller Hinton
agar. Remove excess fluid by pressing and rotating the
swab against the side of the tube above the level of the
suspension.
- Streak the swab evenly over the surface of the
medium in three directions rotating the plate
approximately 60o to ensure even distribution.
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 Cont…..

4. With the Petri dish lid in place, allow 3 – 5 minutes for the
surface of the agar to dry.
5. Using sterile forceps or multi disc dispenser, place the
appropriate antimicrobial discs evenly distributed on the
inoculated plate.

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 Cont….
6. Within 30 minutes of applying the discs, invert the
plate and incubate it aerobically at 35oC for 16 – 18
hours.
7. After over night incubation, examine the control and
the test plates. Using a ruler measure the diameter of
each zone of inhibition in mm on the underside of the
plate. the end point of inhibition is where growth
starts.

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 Cont…
Interpretation of Zone Size
Using the interpretative chart, interpret the zones sizes
of each antimicrobial and report the organisms as
‘Resistant’, Intermediate (moderately sensitive) or
‘Sensitive’ (susceptible).

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 THANK YOU

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