Life Sciences Group

International Journal of Veterinary

Science and Research
DOI: http://dx.doi.org/10.17352/ijvsr CC By

Special Issue: Manual guidance of veterinary clinical practice and laboratory

Abdisa Tagesu*
Research Article
Jimma University, School of Veterinary Medicine,
Jimma, Oromia, Ethiopia
Microbiology examination
Received: 14 May, 2018
Accepted: 13 August, 2018
Published: 14 August, 2018
Whereas, differential staining uses three reagents like primary
*Corresponding author: Abdisa Tagesu, Jimma
University, School of Veterinary Medicine, Jimma, stain, decolourizer and counter stain. The primary satin imparts
Oromia, Ethiopia, Tel: +251933681407, colour to all the cells, the decolourizer is used to establish a
E-mail: colour contrast and counter stains the cells that are decolourised
https://www.peertechz.com [5]. Staining are adding the color to bacterial cell and used to
deterimine bacterial morphology and to distinguish bacteria
from different species by differential staining characteristics.
Veterinary microbiology laboratory examination is required Before staining all slides are fixed by heat, methyle alcohol and
to establish the clinical samples, etiology and line treatment formalin (Figure 3). Fixation is important to make permeable
through antibacterial sensitivity testing. Veterinarian should staining to bacterial cell by killing vegetative bacteria and
have to go for antimicrobial sensitive test before treatment protoplasmic shrinkage [1].
of patient with antibacterial drugs, without testing of
antimicrobial sensitive, the bacteria or microorganism may Gram staining technique
develop resistance to that drugs. In microbiology laboratory
examinations, the clinical samples can be examined in different Gram staining is used in bacteriological to differentiate
ways, the ways are by direct examination and culturing on Gran negative (stain red to pink) from Gram positive (stain
media and isolation of organism. purple to blue) bacteria. Cell wall, aging or death may cause
Gram positive bacteria to appear Gram negative (Figure 1)
Direct examination in microbiology examination [6]. The cell walls of the two groups are morphologically and
chemically quite different. One explanation for the differential
The collected samples can be directely examined are staining reaction emphasizes the higher lipid content of the
tissues, organ swabs (nasal, fecal, vaginal, and prepucial and cell walls in Gram-negative bacteria. During the decolorization
conjuctival), urine, blood and exudate [1]. The sample can
be collected for bacterial culture or direct examination from
different organ and postmortem as it is detailed in table 1.
Table 1: Sample collection for bacterial isolation from sick animals and carcass [2].

Staining and its principle in Microbiology diagnosis

Direct examination of the collected sample can be conducted

by smearing of tissue or organ on clean and dry glass slide. Thin
smear are prepared from blood, exudate and swabs, air dried
and fixed on flame of Bunsen burner for a few seconds. A stain
is a substance that adheres to a cell, giving the cell color. The
presence of color gives the cells significant contrast so they are
much more visible. Staining is an auxiliary technique used in
microscopy to enhance contrast in the microscopic image [3].
The stain can be classified into simple and differential staining
[4]. Simple staining are used to stain negatively charged
particles, usually the bacterial cell wall components and nucleic
acid carrying negative charge which strongly attract the basic
stain with the positively charges chromogen.

The morphology and arrangement of the bacteria are

visualized by simple stainining. The basic stains commonly
used are methylene blue, crystal violet and carbol fuchsin.

065

Citation: Tagesu A (2018) Microbiology Examination. Int J Vet Sci Res s1: 065-077. DOI: http://dx.doi.org/10.17352/ijvsr.s1.110
step, alcohol may extract the lipids, increasing the porosity or
permeability of the cell walls. Thus, the crystal violet-iodine
complex is easily lost.

The Gram-positive bacteria, however, do not have lipid-rich

cell walls. Their cell walls become dehydrated during the alco-
hol treatment, decreasing the porosity so that the crystal vio-
let-iodine complex is retained [7]. Gram-positive organisms
are able to retain the crystal violet stain because of the high
Figure 3: General staining techniques in veterinary microbiology (http://micro-
amount of peptidoglycan in the cell wall. Gram positive cell
beonline.com).
walls typically lack the outer membrane found in Gram-nega-
tive bacteria [8].
The acetone alcohol mixture acts as the decolorizer that
The cell walls of gram positive bacteria are thicker with less washes the stain away from everything in the smear except
lipids substance than gram negative bacteria (Figure 2). The the ram positive organisms. The safranin is the counter stain
solvent dissolves the lipids, which combined with thinner cell that stains everythings in the smear that has been decolorized
walls, washes out or decolorizes the stain. Counter stain (saf- in gram negative organisms [9]. The method of Gram stain is
ranin) is added to exaggerate the contrast with gram negative named after its inventor, the Danish scientist Hans Christian
cells. However, in gram positive bacteria, when washed with Gram1853–1938, who developed the technique while working
solvent, the cell pores close becoming less permeable and are with Carl Friedländer in the morgue of the city hospital in
able to retain the stain and cell become purple. The crystal vio- Berlin in 1884. Gram devised his technique not for the purpose
let is primary stain, which stains everthings in the smear purple of distinguishing one type of bacterium from another but to
make bacteria more visible in stained sections of lung tissue
blue. Gram’s iodine acts as a mordant that causes the crysatal
[10].
violet to penetrate and adhere to the gram positive cell [8].
The following are procedure of gram staining (Figure 1,3)
[2,11,12]:

First of all, presenting all material required for staining,

like staining reagent, bacterial sample, Bunsen burner, slide,
cover slide, water, pipette, loop, forceps.

✓ First sterile the loop and take specimen of bacteria or

swabs in to slide

✓ Fixation of slide with the specimen by passing over heat

(flame) several time us in forceps.

✓ Flood the fixed smear with crstal violet solution and

allow remaining for 60 seconds.

✓ Rinse off the crstal violet with distilled or tap water

✓ Flood the slide with iodine solution and allow remain

for 60 seconds.

✓ Rinse off the iodine solution with distilled or tap water

Figure 1: The procedure of gram staining (http://www.wikiwand.com/en/Gram_ ✓ Flood the slide with decolorize for 5 seconds
stain).
✓ Rinse off the decolorizer with distilled or tap water

✓ Flood the slide with safranin and allow to remains for

30 seconds.

✓ Rinse off the Safranin with distilled or tap water.

✓ Dry the slide on absorbent paper and place in an upright

position.

✓ Put stained slide under microscope at 100 x objectives

and observe the color change of bacterial specimen,
and the gram positive (Stain deep violet to blue) and
Figure 2: Structure of gram positive and negative bacteria (https://bio.libretexts.
negative bacteria (stain pink to red) will be observed.
org).
066

Citation: Tagesu A (2018) Microbiology Examination. Int J Vet Sci Res s1: 065-077. DOI: http://dx.doi.org/10.17352/ijvsr.s1.110
Acid fast staining (The Ziehl–Neelsen stain) ✓ Add additional stain ( carbol fuchsin) and heated stain
remain on slide for 2-5 minute
The outer layer of the mycobacterial cell wall consists
mainly of long-chain fatty acids called mycolic acids [13]. ✓ Wash off the stain with clean water
These mycolic acids comprise approximately 40% of the dry
✓ Cover the smear with 3% acid alcohol or 20% sulfuric
weight of the bacterium and are partially responsible for its
acid for 2-5 minutes, until the smear is sufficiently
acid fastness and the relative impermeability of the cell wall,
decolorized (pale pink).
including impermeability to antibacterial agents [14,15].
✓ Add a bit more decolorizer for very thick slides or to
The Ziehl – Neelsen stain was first described by two
those continue to red dye
German doctors; Franz Ziehl (1859 to 1926), a bacteriologist
and Friedrich Neelsen (1854 to 1894) a pathologist. In this ✓ Wash well with water
type some bacteria resist decolourization by both acid and
alcohol and hence they are referred as acid fast organisms. ✓ Cover the stain with malachite green stain or methylene
This staining technique divides bacteria into two groups for 1-2 minutes
namely acid-fast and non acid-fast [16]. Zhiel neeslen is used
✓ Wash off stain with clean water
to detect mycobacteria, chlymdia, nocardia and brucella spps
[17].Once stain penetrated and combine with the mycolic acid, ✓ Wipe the back of the slide clean, and place it in a
and the cells resist decolorization even when a dilute acid draining rack for smear to air dry
alcohol solution is applied. Therefore organism said to be acid
reisitance or acid fast [18]. ✓ Examine the smear microscopically, using 100x oil
immersion and identify bacterial whether acid fast or
Preparation of reagent before staining [11]: non-acid fast.

✓ Ziehl neelsen carbolfuchsin: Dissolve 3 g basic fuchsin ✓ Interpretation: Acid Fast Bacilli: Red, straight or slightly
in 100 ml 95% ethyl alcohol. Prepare a 5% phenol curved rods, occurring singly or in small groups, may
solution by dissolving 5g phenol in 100 ml distilled appear beaded. Cells: Green (malachite green) or Blue
water. Prepared the Ziel Neelsen carbolfuchsin by (methylene blue). Background material: Green (malachite
mixing 10 ml alcoholic basic and 90 ml 5% phenol green) or Blue (methylene blue). The color is depend on
and allowing the mixture to stand for 24 hr. Filter the the reagent which used in procedure as table 2.
solution prior to use.
Spore staining
✓ Acid alcohol: Mix 2 ml concentrated hydrochloric acid &
Bacterial spores are bacterial mechanism that is in
98 ml 95% ethyl alcohol.
tentionally set in an attempt to secure themselves to the
✓ Methylene blue: Prepare solution of methylene blue adverse effects of the external environment (Figure 4). Spore
by adding 1.5 g powdered methylene blue to 100 ml is extremely resistance to harsh environment and disinfectants
95% ethyl alcohol. Slowly add the alcohol to dissolve [20,21]. Endospore staining is the type of staining to recognize
the powder. Add 30 ml saturated alcoholic solution
for methylene blue to 100ml distilled water and 0.1 Table 2: The interpretation of acid fast staining of bacteria (http://microbeonline
ml 10% potassium hydroxide. Filter and dilute it 1: 20 .com).
with distilled water to prepare the final methylene blue
counterstain.

Staining Procedure

The acid fast staining can be conducted as the following

procedure for bacteria which have mycolic acid. The procedure the presence spore in bacterial vegetative cells and can
is started by applying the primary dye (catbolfuchsin or penetrated wall thickness of spore bacteria. Spores may be
malachite green) and putting the smear to heat. The importance located in the middle of the cell, at the end of the cell, or
of heat is to enhance penetration and retention of the dye into between the end and middle of the cell (Figure 5). Spore shape
the cells of bacteria. The general use of the reagents are like; may also be of diagnostic use. Spores may be spherical or
Acid alcohol is decolourize removes the red stain from bacteria elliptical [22]. There are two major pathogenic spore forming
that are non-acid fast. Acis fast organism retains the red colour genera, Bacillus and Clostridium [23].
since carbolfuchsin is more soluble in the cell wall waxs then
Procedure of spore staining (Figure 6) [24,25]:
acidic alcohol. But, in non-acid fast bacteria cell wall lack of
wax components and carbolfuchsin remove rapidily and cell ✓ Prepare a smear of the acid fast species of bacteria and
become colorless [11,17,19]: make it air dry and heat fix.

✓ Allow the film to air dry and then gently heat and fix it. ✓ Put a beaker of water on the hot plate and boil until
067

Citation: Tagesu A (2018) Microbiology Examination. Int J Vet Sci Res s1: 065-077. DOI: http://dx.doi.org/10.17352/ijvsr.s1.110
 steam is coming up from the water. Then turn the hot
plate down so that the water is barely boiling.

✓ Place the wire stain rack over the beaker which now has
steam coming up from the boiled water.

✓ Cut a small piece of paper towel and place it on top of

the smear on the slide. The towel will keep the dye from
evaporating too quickly, thereby giving more contact
time between the dye and the bacterial walls.

✓ Flood the smear with the primary dye, malachite green

or carbonfuchsin, and leave for 5 minutes. Keep the
paper towel moist with the malachite green. Do not let
the dye dry on the towel.

✓ Remove and discard the small paper towel piece.

✓ Wash really well with water and move the slide and wire
rack from the boiling

✓ Water to the regular stain tray to finish up the last step

in the procedure. Figure 6: Procedure of spore staining. http://laboratoryinfo.com and http://
microbiologyinfo.com.
✓ Place the smear in the stain jar or flood the smear with
the counterstain dye, safrinin, and leave for 30 seconds-
60 seconds. ✓ Wash well with water. Blot dry with bibulous paper.

✓ Examine the slide under microscope for the presence of

endospores. Endospores are bright green and vegetative
cells are brownish red to pink which picked up form
safranin dye. The spore can be one terminal (Cl. Tetani),
central to terminal (Bacillus anthracis) and subterminal
(Cl.perfringe) location.

Capsule staining

The capsule stain is a type of differential stain which

selectively stains bacterial capsules. A capsule is a substance
that is synthesized in the cytoplasm and secreted to the outside
of the cell where it surrounds the bacterium. Capsules can be
polysaccharide, polypeptide, or glycoprotein. Capsules are
associated with virulence in several microorganisms, including
Streptococcus pneumonia and Neisseria meningitidis, because
capsules provide a mechanism for these pathogens to evade
the host immune system. Because of their structure and
composition, heat and water will dislodge capsules from
bacteria during laboratory procedures. In the capsule staining
procedure, the primary stainis crystal violet, and all parts of
the cell take up the purple crystal violet stain. There is no
mordant in the capsule staining procedure. A 20% copper
Figure 4: Sporulation of bacteria while change from unfavorble condition. htpp sulfate solution serves a dual role as both the decolorizing
://commons.wikimwdia.org-wiki-File :Sporulation_in_Bacillus_subtilis.jpg, http:// agent and counter stain. It decolorizes the capsule by washing
online.science.psu.edu-micrb106_wd/node/6057. out the crystal violet, but will not decolorize the cell. As the
copper sulfate decolorizes the capsule, it also counter stains
thecapsule. Thus, the capsule appears as a faint blue halo
around a purple cell [26].

Procedure of capsule staining [27]:

✓ Obtain a clean glass slide. Choose one of the above broth

cultures, and agitate your broth culture to disperse the
Figure 5: Morphology of endospores : Terminal(a,d,e); Subterminal (b) ; Central(c,f)
bacteria.
; circular(b,d), ellipsoid(a,c,e,f).http://ttktamop.elte.hu.
068

Citation: Tagesu A (2018) Microbiology Examination. Int J Vet Sci Res s1: 065-077. DOI: http://dx.doi.org/10.17352/ijvsr.s1.110
 ✓ Prepare a smear using 2-3 loopfuls of the broth culture. is used to grow gram negative bacteria and to identify those
Allow the smear to air-dry, but do not heat fix this slide! which ferment lactose and also differential, meaning that
Heat will cause the capsule to dislodge. this medium differentiates or distinguishes between groups
of bacteria on the basis of a color change reaction. MacCon-
✓ Cover the smear with crystal violet, and stain the smear key’s contains two additives that make it differential; neu-
for 2 minutes. tral red (a PH indicator) and lactose (a disaccharide) [29-31].

✓ Tilt the slide and rinse with 20% copper sulfate solution. MacConkey Agar media contains crystal violet and bile
Do not rinse with water! Water will remove the capsule salts, which inhibit most gram-positive organisms and select
from the cell. for gram-negative organisms (Figure 8). It also contains the
substrate lactose and the pH indicator neutral red, which allow
✓ Let the slide air dry for a few minutes. Do not blot the
differentiation among gram-negative bacteria based on their
slide! Blotting will remove the bacteria from the slide
ability to ferment lactose. If an organism is unable to ferment
and/or distort the capsule.
lactose, the colonies will be colorless, taking on the color of the
✓ Observe the slide under oil immersion, and Look for medium.
purple cells surrounded by a clear or faint blue halo on
Lactose fermenter bacteria are like Citrobacter spp, Klebsi-
a purple background. (The halo is the capsule.) You may
ellaspp, Escherichia coli, and Serratia spp. and lactose fermen-
need to decrease the amount of light in order to make
ter are like Proteus spp, Shigella spp, Yersinia spp, Salmonel-
the capsule easier to see.
la spp, Edwardsiella spp, Hafnia spp, Morganella spp, Providencia
✓ Clean your microscope with lens cleaner, removing spp and Gram positive bacteria are nor growth on mackonkey
[32,33]. Growth on MacConkey agar indicates the organism
all oil from lenses and the capsule will be observed as
is resistant to crystal violet and bile salts, and is likely to be
figure 7.
gram-negative. Enteric bacteria that have the ability to fer-
Cultural methods ment lactose can be detected using the carbohydrate lactose,
and the pH indicator neutral red. Growth which is a pinkish-
The purposes of culturing media in veterinary clinical red color indicates the organism has the ability to ferment lac-
samples are to cultivate the organism and to obtain the tose [33].
discrete colonies for isolation of organism in the pure culture.
The cultural media which used in veterinary laboratory are
classified in to four groups, like simple, enriched, selective and
differential or biochemical media [26,28].

Simple media: It is also called general media or universal

media. It is a media which used for bacteriological examinations
of the clinical specimens and support the microorganisms that
do not require special nutrients. Example: nutrient broth,
nutrient agar.

Enriched media: The cultured media that enriched with

whole blood, serum, vitamins, special extraction which
support for growth of fastidious organisms. Example: Blood
agar, serumagar, chocholate agar. Figure 7: Capsule staining. Capsule straining is neggative staining techniques
due to its stain only bacterial cell and background, but capsule stay in unstained
Selective media: It allows the growth of certain types of around bacterrial cell [27].
organisms and prevents or slows down growth of bacteria
other than pathogens for which media are intended. Example:
MacConkey agar, Salmonella shigella agar.

Biochemical or differential media: It is the media that used

to identification of one bacterial species from other.The media
which indicator substances are added to differentiate bacteria.
Example: TCBs agar differentiates sucrose ferment to non-
sucrose fermenter. The routine bacteriological examinations
are carried out by using blood agar or tryptose soya agar plates/
McConkey agar plates. Example: mannitol salt agar, DNAse
agar, blood agar, MacConkey agar and eosin-methylene blue
agar.

MacConkey Agar is both selective and differential [29-

31]. MacConkey agar is a selective culture medium, which Figure 8: Macconkey Agar : (http://chrysacsiovi.tk).

069

Citation: Tagesu A (2018) Microbiology Examination. Int J Vet Sci Res s1: 065-077. DOI: http://dx.doi.org/10.17352/ijvsr.s1.110
 Eosin Methylene Blue (EMB) agar is both selective and dif- ● Alpha hemolysis: when bacteria causes partial lysis
ferentia. Eosin methylene blue is selective for gram negative of red blood cells and hemoglobin, which results in a
bacteria, the dyes eosin and methylene blue found in medium greenish-grey or greenish-yellow discoloration of the
which inhibit growth of gram positive bacteria [33,34]. It also blood around the colonies.
contains lactose, allowing differentiation between organisms
● Gamma hemolysis: when the bacteria couldn’t causes
which ferment lactose and produce acid end-products, and or-
the hemolysis of the red blood cells and hemoglobin.
ganisms that do not ferment lactose. Small amounts of acid
Then no any change in the medium.
production result in a pink colored growth (e.g. Enterobacter
aerogenes) while large amounts of acid cause the acid to precip- Culture transfer techniques
itate on the colony, resulting in a characteristic greenish, me-
tallic sheen (e.g. E. coli). Organisms which do not ferment lac- Various media types used in microbiology labs include agar
tose will be colorless, taking on the color of the medium [34]. slants, agar deeps, agar plates, and broths. An agar slant is a
solid media in a test tube with a slanted surface on which to
Blue agar indicates the organism can grow in the presence culture the microorganism. These are typically inoculated by
of the dyes eosin and methylene blue and is likely a gram- streaking the surface of the slant with a sterile loop. An agar
negative. Growth which is a pink color indicates the organism deep is a solid media in a test tube which does not have a
can ferment lactose to form weak acid end-products, and slanted surface. These are typically inoculated by stabbing the
growth which exhibits a green metallic sheen indicates the media with a sterile needle. An agar plate is a solid media which
organism can ferment lactose to form strong acid end-products is contained in a Petri plate, providing an optimal surface on
[35] (Figure 9). which to culture microorganisms. Like the agar slants, these
are inoculated by streaking the surface with a sterile loop.
Mannitol salt agar (7.5% NaCl) is a medium selective for
Broth tubes are a liquid medium which can be inoculated by a
staphylococci and differential with respect to mannitol
sterile loop, needle, or pipette [26].
fermentation. It contains a high concentration (about 7.5%-
10%) of salt (NaCl), making it selective for Gram-positive
bacteria (Staphylococcus and Micrococcaceae) since this level of
salt is inhibitory to most other bacteria. Staphylococcus aureus
produces yellow colonies with yellow zones, whereas other
coagulase-negative staphylococci produce small pink or red
colonies with no colour change to the medium .If an organism
can ferment mannitol, an acidic byproduct is formed that
causes the phenol red in the agar to turn yellow [36].

Fermentation of mannitol is only seen in the pathogenic

species of Staphylococcos and is signaled by the production of
acidic products leading phenol red in the media to change
Figure 9: The eosin methylene blue. Quadrant 1 : Growth on the plate indicates
from a neutral red-orange to bright yellow. Non-pathogenic the organism, Escherichia coli, is not inhibited by eosin and methylene blue and
staphylococci produce small colonies surrounded by red or is gram-negatuive bacterium. The green metallic sheen indicates E.coli is able to
pink- purple zones due to the production of basic by products ferment lactose to produce straon acid end-products. Quadrant 2 : Growth on the
due to metabolism36,37.A change in the color of the agar from plate indicates the organism, P seudomonas aeruginosa, is not inhibited by eosin
and methylene blue and is a gram-negative bacterium. The absence of color in the
pink to yellow indicates the organism has the ability to ferment
bacterial growth indicates P. aeruginosa is unable to ferment lactose. Quadrant
mannitol. Yellow coloration can usually be seen around the 3 : Growth on the plate indicates the organisam, Enterbacter aerogenes, is not
sides of the bacterial growth [38]. inhibited by eosin and methylene blue and is a gram-negative bacterium. The pink
color of the bacterial growth indicates E. aerogens is able to ferment lactose to
Blood Plates: All organisms could growth on the blood produce weak acid end-products. Quadrant 4 : Absence of growth indicates the
plates, but they are not selective. Blood agar is a rich medi- orgganism, staphylococcus aureus, is inhibited by eosin and methylene blue and
um that has been supplemented with fresh 5-10% blood. The is a gram positive bacterium.
(http://iws2.collin,edu/dcain/CCCCD%20Micro/embagar.htm).
hemolytic response can be dependent upon the type of blood.
Sheep blood is commonly used, but some organisms require
rabbit or bovine blood [39,40]. Blood agar is a differential me-
dium. It is also commonly used as an enriched medium for
growing fastidious bacteria. Some bacteria produce exotoxins
called hemolysins that cause lysis of red blood cells.

The degree of the hemolysis is an especially use-

ful tool for differentiation among Gram-positive coc-
ci. The three types of hemolysis are (Figure 10) [39,39]:

● Beta hemolysis: When the bacteria cause complete

lysis of red blood cells and hemoglobin, this results in
complete clearing of the blood around colonies. Figure 10: Types of heamolysis on blood agar.

070

Citation: Tagesu A (2018) Microbiology Examination. Int J Vet Sci Res s1: 065-077. DOI: http://dx.doi.org/10.17352/ijvsr.s1.110
 Procedure

✓ Label each tube appropriately.

✓ Using a sterile needle, obtain a small amount of culture

from the broth tube containing

✓ Use the needle to inoculate a nutrient agar deep tube by

stabbing the needle into the agar deep.

✓ Using a sterile loop, obtain a loop full of culture from

the broth tube containing cultured bacteria. Use the loop
to inoculate a nutrient agar slant tube by streaking the Figure 11: Four streaking techniques in isolation and inoculating method of
agar slant with a zig-zag motion. bacteria.

✓ Using a sterile loop, obtain a loop full of culture from

the slant of cultured bacteria  Tape plate closed on both sides. Make sure the plate is
labeled with your name, date, and the organism(s), and
✓ Use the loop to inoculate a nutrient broth tube by gently incubate upside down (to prevent condensation from
swishing the loop around in the liquid broth. getting on to agar) at 30 °C.

✓ Make sure all caps are loose, but secure. Serial dilution

✓ Incubate at 30 °C for 48 hours. The purpose of serial dilution in veterinary microbiology

laboratory is to quantifying the number of bacteria in a broth
Isolation of pure colony culture and also necessary to quantify the number of living
bacteria in a particular sample. A Pure culture may be obtained
A colony forms on a plate when a single microbe is
by serially diluting the sample with sterile water to the point of
inoculated onto the surface of the plate and reproduces until
extinction in number of cells. This method is used to isolate the
there are enough cells to form a visible colony. Since a colony
organisms, if it is present in large number in the mixture [42].
theoretically forms from a single cell, a colony should then
The liquid culture needs to be diluted, often 1-million-fold,
represent a pure culture. One way to obtain single, isolated
before it can be plated. When such a large dilution is required,
colonies is using the quadrant streak method [41]. The quadrant
an accurate dilution cannot be made in a single dilution step
streak plate method allows sequential dilution of the original and it is necessary to make serial dilutions. Serial dilutions
microbial material over the entire surface of a fresh plate are a step-wise set ofdilutions which sequentially dilute the
(Figure 11). After the original sample is diluted by streaking bacterial culture. One or more of the dilutions are then plated
it over successive quadrants, the number of organisms will be on the agar plates to determine the number of colonies present
decreased. Usually by the third or fourth quadrant only a few in the original culture.
organisms are transferred, and these produce single, discrete
colonies. Only plates containing between 30 and 300 colonies are
counted to ensure statistically significant data. To estimate
Procedure [41-143]: the number of bacterial in the original culture, the number of
colonies on the plate is multiplied by the total dilution plated.
 Label different nutrient agar plates.
For example, suppose 0.1 ml of a 10-6 dilution was plated, and
123 colonies were counted following incubation. The total
 Divide the agar plate into 4 quadrants.
dilution plated would be 10-7(since only 0.1 ml was plated),
 Place a loopful of culture onto the plate in Quadrant 1 and the number of bacteria/ml of the original culture would
with a sterile loop and streak the loop very gently using be: (123) x 1/10-7= 1.23 x 109 CFU/ml. Note that the results are
a back and forth motion. expressed as “colony forming units (CFU)” per ml [42,43]
(Figure 12).
 Sterilize loop. Go back to the edge of Quadrant 1 and
extend the streaks into Quadrant 2, going back into Calculate the number of bacteria (colony forming units) per
Quadrant 1 twice. milliliter or grams of sample by dividing number of colonies by
dilution factor multiplied by amount of specimen added to agar
 Sterilize loop. Go back to the edge of Quadrant 2 and plate, the equation can be:
extend the streaks into Quadrant 3, going back into N
Quadrant 2 twice. C=
S* D
 Sterilize loop. Go back to the edge of Quadrant 3 and
æ CFU ö÷
C = concentration çç
extend the streaks into Quadrant 4, going back into çè mL ÷÷ø
Quadrant 3 twice. Be careful NOT to go back into
Quadrant 1! N = Numbers of colonies
071

Citation: Tagesu A (2018) Microbiology Examination. Int J Vet Sci Res s1: 065-077. DOI: http://dx.doi.org/10.17352/ijvsr.s1.110
 ✓ Mesophile: Those bacteria grow at ranging from 20
to 45 oC. Example, Staphylococcus aureus (45oC) and
Staphylococcus epidermidis (40oC).

✓ Thermophile: Those bacteria grows at ranging from 45

to 80 oC

✓ Hyperthermophile: Those bacteria grows at ranging

from 80 to 105 oC

✓ Bacterial growth is also dependent on the presence

of oxygen in the environment, as different bacteria
Figure 12: Serial dilution methods in bacterial isolation.
have different oxygen requirements depending on the
types of enzymes they possess. The major bacterial
D = Dilluitions blank factor oxygen classes are aerobes, microaerophiles, obligate
anaerobes, aerotolerant anaerobes, and facultative
S = Volume transferred to plate anaerobes [44,45] (Figure 13).

Procedure how to dilute [43]: • Aerobes require atmospheric O2 (20%), and use O2 as
the final electron acceptor in the electron transport
✓ Prepare serial dilutions of the broth culture as shown system.
below. Be sure to mix the nutrient broth tubes before
each serial transfer. Transfer 0.1 ml of the finalbthree • Microaerophiles require O2 at below atmospheric
dilutions (10 ,10-5 -6
,10 -7)
to each of three nutrient agar concentrations, typically 2-10%. Microaerophiles have
plates, and label the plates. a limited ability to neutralize toxic oxygen, so excess O2
will kill the bacteria. However, microaerophiles do use
✓ Position the beaker of alcohol containing the glass O2 as final electron acceptor in the electron transport
spreader away from the flame. Remove the spreader system. Example: Campylobacter jejuni, Helicobacter
and very carefully pass it over the flame just once (lab pylori.
instructor will demonstrate). This will ignite the excess
• Obligate Anaerobes cannot survive in the presence
alcohol on the spreader and effectively sterilize it.
of any oxygen. Obligate anaerobes lack the enzymes
✓ Spread the 0.1 ml inoculum evenly over the entire necessary to break down the toxic by-products of
surface of one of the nutrient agar plates until the oxygen. Examples are like: Clostridium, Bacteroides.
medium no longer appears moist. Return the spreader
• Aero tolerant: Anaerobes grow equally well in the
to the alcohol.
presence or absence of oxygen. They do possess enzymes
✓ Repeat the flaming and spreading for each of the necessary to neutralize toxic oxygen by-products, but
remaining two plates.

✓ Invert the three plates and incubate at room temperature

until the next lab period.

The effective on growth of bacteria

Bacterial growth is depending on the temperature may

affect bacterial enzymes, membrane fluidity. The enzymes
are less active at low temperatures due to low kinetic energy
and increasingly as temperature increase. However, when
temperature becomes too high, enzymes become denatured
and will cease its functions. At low temperatures, the lipids ina
cell membrane can pack too tightly and solidify this prohibiting
memebrane from proper functions. When temperatures become
excessively high, the lipid bilayer can become too fluid and lose
its integrity. Bacteria can be divided into four depending on
ability to survive in temperature environment; all prokaryotes
can be classified into four general groups depending on their
temperature requirements [44].

✓ Psychrophile: Those bacteria grows at ranging from -5

to 20 oC Figure 13: Classification of bacteria depending on their environment (requirement
of oxygen).
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Citation: Tagesu A (2018) Microbiology Examination. Int J Vet Sci Res s1: 065-077. DOI: http://dx.doi.org/10.17352/ijvsr.s1.110
 they never use O2 as a final electron acceptor. Example:
Enterococcus faecalis, some Lactobacillus

• Facultative Anaerobes are also able to live either in the

presence or absence of oxygen, but they prefer oxygen
so they can carry out aerobic respiration with O2 as
final electron acceptor to maximize ATP yields. These
organisms can use other electron acceptors if O2 is
not available, such as fumarate and nitrate. They can
also utilize fermentative metabolism in the absence of
oxygen. Example: All Enterobacteriaceae (E.coli), some
Bacillus, Staphylococcus aureus.

• Obligate aerobic bacteria: They have respiratory

enzymes and lack the capacity for fermentations.
Example: Pseudomonas, some Bacillus, Mycobacterium
tuberculosis

• Capnophilic bacteria require increased concentration

of carbondioxide (5% to 10%) and approximately 15%
Figure 14: Creating anaerobic envitonment in culturing of bacteria by using jar
oxygen. This condition can be achieved by a candle jar
and candle jar.
(3% carbondioxide) or carbondioxide incubator, jar or
bags. (Haemophilus influenza, Neisseria gonorrhoeae).
generic level, some of the tests can be carried out to identify
Cultivation of bacteria on anaerobic condition the species. Secondary biochemical tests are: Fermentation of
carbohydrates, Citrate utilization, Decarboxylation of amino
Specialized methods are necessary to culture organisms
acids, gelatin liquefaction, hydrogen sulfide test, indole test,
anaerobically. One such method is the use of fluid thioglycollate
Methyl red test, nitrate reduction test, ONPG test, Urease test
broth, which is a reducing medium. It contains sodium
and Voges Proskauer test [46]. The bacteria generally classified
thioglycollate, which reacts with molecular oxygen keeping
based on their biomedical test and staining and morphological
free oxygen levels low. The sodium thioglycollate in the broth
structure is detailed in figure 15.
creates a redox potential in the tube, with higher levels of
oxygen at the top of the tube, and a complete absence of oxygen Catalase test
at the bottom of the tube. Fluid thioglycollate broth also
typically contains a redox potential indicator such as resazurin, Catalase is the enzyme which present in most cytochrome
which produces a pink coloration in an oxidized environment. containing aerobic and facultative anaerobic bacteria used to
break hydrogen peoxide in to Oxygen (O2) and water (H2o).
A second method used to culture organisms anaerobically When the hydrogen peroxide is added to bacterial sample, it
is the use of a GasPak Jar and candle method [45] (Figure produces some bubble. When bubble is formed it shows, that
14). This is a specialized culture vessel in which an anaerobic bacteria is catalase posistive [47] (Figure 16).
environment is generated after inoculated media are sealed
into the chamber. Anaerobic conditions are created by adding ✓ Catalase positive:
water to a gas generator envelope that is placed in the jar
✓ Staphylococcus aureus,
just before sealing. There are two chemical tablets in the
envelope, sodiumborohydride and sodium bicarbonate. Water ✓ Micrococcus,
reacts with these chemicals, producing hydrogen gas from
the sodium borohydride and carbon dioxide from the sodium ✓ Bacillus,
bicarbonate. The hydrogen gas combines with free oxygen in
✓ Listeria monocytogenes,
the chamber to produce water, thus removing all free oxygen
from the chamber. This reaction is catalyzed by the element ✓ Enterobacteriacae,
palladium, which is attached to the underside of the lid of the
jar. The carbon dioxide replaces the removed oxygen, creating ✓ Gonococcus&Meningococcus,
a completely anaerobic environment [45].
✓ Vibriocholerae,
Biochemical test
✓ Pseudo/Aero/Plesiomonas,
There are two levels of biochemical tests. The Primary
Identification test in which, once a pure culture is obtained, the ✓ Catalase negative:
results from a few comparatively simple tests can often identify
✓ Streptococcus spp
the bacterium to a generic level. The Secondary Identification
of Bacteria occurs once the bacterium has been identified to a ✓ Clostridium

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Citation: Tagesu A (2018) Microbiology Examination. Int J Vet Sci Res s1: 065-077. DOI: http://dx.doi.org/10.17352/ijvsr.s1.110
Triple Sugar Iron Agars test

Triple Sugar Iron Agars (TSI) is used to determine if bacteria

can ferment glucose and/or lactose and if they can produce
hydrogen sulfide or other gases (Figure 17) [48]. This method
used to differentiate various Enterobacteriacae, including
Salmonella and Shigella, which are intestinal pathogens. TSI
contains three sugars: glucose, lactose and sucrose. Lactose
and sucrose occur in 10 times the concentration of glucose
(1.0% versus 0.1%). Ferrous sulfate, phenol red (a pH indicator Figure 17: Triple sugar test and its interpretation.
that is yellow below pH 6.8 and red above it), and nutrient agar
are also present. The tube is inoculated by stabbing into the
sodium chloride, 0.3 gram of sodium thiosulphate, 0.024 gram
agar butt (bottom of the tube) with an inoculating wire and
of phenol red and 12 gram of agar and make the mixture up to
then streaking the slant in a wavy pattern. Results are read at
1000ml with distilled water. The Peptone mixture and the Beef
18 to 24 hours of incubation [38,49,50].
and Yeast extracts provide the nutrients essential for growth.
Procedure: Sodium chloride maintains the osmotic balance of the medium.
The Bacteriological agar is the solidifying agent.
Preparation of media: Add 3.0 gram of Beef extract, 3 gram
of yeast extract, 15 gram of peptone, 5 grams of protease The procedures are performed as below [51]:
peptone, 10.0 grams of lactose, 10.0 gram of saccharose, 1.0
 Sterilize the inoculating needle in the blue flame of the
gram of glucose, 0.2 gram of ferrous sulphate, 5.0 gram of
bunsen burner till red hot and then allowed to cool.

 From the rack, take the Trypticase soy broth tube

containing the 24-48 hour culture, remove the cap and
flame the neck of the tube.

 Using aseptic technique, take the culture of the organism

from the TSB (tryptic soy broth) tube with the needle.

 Again flame the neck of the tube and replace the tube in
the test tube rack.

 Take a sterile TSI slant tube from the rack, remove the
cap and flame the neck of the tube.

 Stab the needle containing the pure culture into the

medium, upto the butt of the TSI tube, and then streak
the needle back and forth along the surface of the slant.

 Again flame the neck of the TSI tube, cap it and place it
in the test tube rack.

 Incubate at 37oc for 18 to 24 hours.

Interpretation:
Figure 15: Classification of bacteria depending on their morphological biochemistry
(www.afrivip.org).
 A yellow slant on TSI indicates the organism ferments
sucrose and/or lactose.

 A yellow butt shows that the organism fermented

glucose.

 Black preciptate in the butt indicates hydrogen sulfide

production (Salmonella typhi).

 Production of gases other than hydrogen sufide is

indicated either by cracks or bubbles in the media or the
media being pushed away from the bottom of the tube.

Urease test

Figure 16: Catalase test and its interpretation. Urease test is used to differentiate urease positive proteuis

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Citation: Tagesu A (2018) Microbiology Examination. Int J Vet Sci Res s1: 065-077. DOI: http://dx.doi.org/10.17352/ijvsr.s1.110
species from other members of enterobacteriacea. Some species
of bacteria possess the enzyme urease and able to hydrolyze
urea with the release of ammonia and carbon dioxide. Urea
broth is a yellow orange color. The enzyme urease will be used
to hydrolyse urea to make ammonia. If ammonia is made, the
broth turns a bright pink color and it is positive and in negative
result no color change. The result of interpretation positive test
is magenta to pink colour (Figure 18) [38,52].

Motility test

Major bacteria are motile by flagella. Motility can be

temperature dependent and some bacteria tend to be motile
at ambient temperature but not at 37 o c. semisolid motility
media are important with Terazolium salts can be added to
these media aid in detection of motility. Before autoclaving the Figure 18: Urease test and its interpretation.
motility medium, 0.05 g of 2, 3, 5-triphenylterazolium chloride
is added per litre of medium. Teratozolim is colorless, but a
bacterium is grow the dye is incorporated in to bacterial cells
where it is reduced to an insoluble red pigment, formazan.
The red colour forms only in the area of medium where the
bacterium is growing. Motility media are prepared in test
tube, two test tube of the medium are stab- inoculated using
a straight wire. One tube is incubated at room temperature
and the other at 37oC. The tube are examined for motility after
Figure 19: Motility test medium tubes containing TTC inoculated with escherichia
24 and 48 hrs. Motile bacteria migrate through semisolid
coli (a) and bacillus cereus (b). Positive before treatment negative after treatment
medium which become turbidity. If TTC has been incorporate with prodigiosin [53].
into the medium, the motility is demonstrated by a red colout
throughout the agar. The growth of non-motile bacteria is
confined to the stab line [53] (Figure 19).

Coagulase Test

The coagulase test usually relates with pathogenicity, some

staphylococci can be negative to the slide coagulase test, but
positive to the tube test.

Slide coagulase test: Heavy loopful of the staphtlococci

culture is emulsified in a drop of water on microsope slide. A
loop of rabbit plasma is added and mixed well with the bacterial
Figure 20: Coagulase test by two methods.
suspecntion. The slide is gently rocked and a positive reaction
is indicated by clumping within 10 seconds (Figure 20).

Tube coagulase test: 0.5 ml of rabbit plasma is placed in

a small test tube. A suspension (0.01) of an overnight brothy
culture is added to the rabbit plasma test tube. The tube rotated
gently to mix the contents and then incubated at 35-37OC,
preferably in a water bath. Alternatively one to three large
well isolated colonies can be transferred into 0.05 ml of rabbit
plasma in a tube and incubated at 35-37OC a positive with
degree of clotting of the plasma can occur in 2- 4hours.many
weak coagulase positive strains will coagulate at the plasma
only after overnight incubation [17,38] (Figure 20) . Figure 21: Citrate test to differentiate enterobactereciae spps. A : Citrate negative
: No groth and no color change will be visible, the medium remain the deep forest
Citrate utilization test green color of uninoculated agar, Only bacteria that can utilize citrate as the sole
carbon and energy source will be able to grow on the simmons citrate medium,
Citrate utilization test is commonly employed as part thus a citrate-negtive test culture will be virtually indistinguishable from an
of a group of tests, the IMViC (Indole, Methyl Red, VP and uninoculated slant. B : Citrate positive : Growth of bacteria will be visible on the
Citrate) tests, that distinguish between members of the slant and medium will be an intense prissian blue. The alkaline carbonates and
bicarbonates produced as by-products of citrate catabolism rasise the pH of the
Enterobacteriaceae family based on their metabolic by-
medium to above 7.6, causing the bromothymol blue to change from the original
products [54]. green clolor to blue.
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Citation: Tagesu A (2018) Microbiology Examination. Int J Vet Sci Res s1: 065-077. DOI: http://dx.doi.org/10.17352/ijvsr.s1.110
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Citation: Tagesu A (2018) Microbiology Examination. Int J Vet Sci Res s1: 065-077. DOI: http://dx.doi.org/10.17352/ijvsr.s1.110