Unit: 6

Microbiology
A. Objectives

 To be familiar about the technique of Gram staining.

 To be familiar with preparation of blood smear.

 To be familiar with preparation of media

 To be familiar with sample collection for bacteriology.

 To be familiar with inoculation method

B. Content elaboration

6.1 Preparations of Media

For the growth of the bacteria different types of media should be prepared on the lab.

Most of the Medias are found prepared in market

1. Nutrient broth: weight the required amount of the media dissolve it with mild heat, after
the media gets dissolved keep it on test tube or bottle and seal it. Keep it on autoclave
and maintain the temperature of 121 ° C for 15-20 minutes. After that take the media out
and refrigerate it at 2-8 ° C.
2. Nutrient agar: weigh the required amount of the media and dissolve it with the help of
mild heat, maintain the PH of 7.4 and autoclave it at 121 °C for 15-20 minutes. After it
gets cooled Keep about 20 ml of media on sterilized Petri Dish and cover it. Leave the
media for about 15-20 minutes on room temperature and let it to fix. It can be stored
for several months at the temperature of 2-8 ° C.
3. MacConkey’s agar: while performing the culture, lactose fermenting bacteria produce
pink colony on macConkey’s agar whereas non lactose fermenting bacteria show no
change in color. Required amount of media is weighed and autoclaved. About 20 ml
of media is taken out from autoclave and kept on Petri Dish and covered. Media can
be prepared on sterilized tube or vial.
4. Blood agar: This is red colored and mostly used for the growth of gram positive
lactose fermenting bacteria. Hemolysis is seen during bacterial growth on blood agar.
Required amount of media is weighed, heated and dissolved. It is autoclaved at 121 °C
for 15-20 minutes. It is brought out from autoclave and cooled at 45-50 ° C. This task
can also be
 performed at water bath. Sterilized blood of sheep/rabbit (5-10%) of total media is added
and cooled slowly. The added blood should not contain any air bubbles. Wait for 15-20
minutes and let it fix at room temperature. The surface of media in Petri Dish should be
leveled to 4 mm and refrigerated. To analyze the contamination it should be kept on
room temperature for 1 day.
5. Chocolate agar: it is chocolate colored agar. This media is used for the growth of
delicate bacteria. While preparing this media blood agar is hemolysed and heated until
chocolate color appears. It is used for the growth of gonococcus, nemococcus and
hemophilus bacteria.

Reference: veterinary prayogshala gyan by Dr. jibaccha shah

6.2 Sample collection for bacteriology

Selection, Collection and Shipping of Specimens

The success and ultimate value of examining clinical specimens bacteriologically are influenced
initially by the care exercised in the selection, collection and shipment of the specimens.
Specimens selected should be those likely to yield the causative agent(s) and efforts should be
made to avoid contamination with organisms from the surrounding environment.

Bacterial Isolation Specimens

 Tissues and organs—Suitable portions of each specimen should be placed in individual

polyethylene bags or other suitable leakproof container. Portions of the intestine (tied off if
appropriate) should be packaged separately.
 Swabs—Swabs may be the preferred way for submitting specimens from nasal
passages, pharynx, tonsil, eye, ear, skin, abscesses, vagina, cervix, etc. Drying of
specimens after collection and during shipment must be avoided by using a suitable
transport medium. Swabs in transport medium are available commercially.
 Feces—Preferably, fecal specimens should be obtained directly from the rectum in a manner
that avoids contamination, placed in a leakproof container, and immediately shipped to the
laboratory. Because of likely contamination, submission of fresh droppings should be
avoided if possible.
 Milk— See section on milk culture for specific sample guidelines
 Urine—Urine must be collected aseptically and refrigerated immediately. Free catch urine
samples should be avoided if possible to avoid contamination. Better recovery of
organisms is achieved from 1-5 ml of urine. Urine swabs are not recommended.
 Brain— Place entire brain in a polyethylene bag or other leakproof container.
 Equine cervical or uterine swabs—Sterile swabs with long handles, referred to as
―disposable guarded culture instruments‖ are available commercially. These instruments do
 not contain a transport media and if there is going to be a time delay, a transport media
should be used.

Disease Requiring Special Consideration

 Anthrax—Cotton swabs are soaked in extruded blood or blood taken from a superficial ear
vein in acute or paracute cases. In swine, swabs should be taken from exudates and cut
surfaces of hemorrhagic lymph nodes.
 Blackleg, malignant edema, etc.—Submission of fresh affected tissues is absolutely essential
since Clostridia from the gut rapidly invades tissues after death. Two (2) impression smears
on glass slides should be included with the submission.
 Clostridial enterotoxemia—Several ounces of fresh intestinal contents or a tied-off section
of affected intestine should be submitted to the laboratory as soon as possible.
 Campylobacteriosis—Bovine and ovine—Semen, preputial washings, fetal stomach
contents, and cervical mucus should be obtained as aseptically as possible and delivered
to the laboratory under refrigeration within five to six hours of collection. Alternatively,
specimens may be frozen as soon as collected with dry ice and shipped in an insulated
container with an adequate amount of dry ice.
 Brucellosis—Placenta, stomach content or aborted fetus, mammary lymph nodes, milk
samples are the preferred specimens for the isolation of Brucella organisms.
Specimens should be shipped to the diagnostic laboratory under refrigerated
conditions.
 Listeriosis—Submit brain stem sample and indicate Listeria is suspected.

6.3 inoculations of media from various types of specimen

Generally 2 methods of inoculation of media are done.
1. Plate culture
2. Testube culture
1. Plate culture: Solid state of media like nutrient agar,blood agar and macConkey
agar prepared in Petri Dish are incubated at 37 °C for 10 minutes to dry the moisture
content on the Petri Dish
Inoculating method:
 Heat the inoculating loop on the Bunsen burner until gets red.
 Dip the inoculating loop on the sample and stab the inoculating loop (3 -4
lines) on the media as shown in the figure.
  Again heat the inoculating loop till it gets red let it cool and again stab the
inoculating loop (3-4lines) on the media from another corner as shown in
the figure.

 Again heat the inoculating loop till it gets red let it cool and again stab the
inoculating loop (3-4lines) on the media from another corner as shown in
the figure.

 Label the Medias and incubate it at 37 °C on incubator for 24 -48 hours.

 After 24-48 hours examine the bacterial colonies present on the Medias. If
the sample is of lungs, liver, heart or any tissues cut it with the help of
sterilized scissors. The liquid obtained while cutting the samples should be
again inoculated with the help of inoculating loop.

Inoculation of culture on test tube

1. Slope method
2. Inoculation on broth
1. Slope method(inoculating on slope media):

In this method straight inoculating needle is used.

 Heat the straight inoculating needle on the Bunsen burner until it gets red
 When the needle gets cool stab a vertical line on the slope media.
 Inoculate zigzag line with the help of inoculating loop on the vertical line stabbed by
the inoculating wire.
 2. Inoculation on broth (liquid) media
 If the sample to be inoculated is on liquid state then inoculation should be done with
the help of sterilized Pasteur pipette on broth or other liquid media.
 If Pasteur pipette is not available then inoculating loop should be used for inoculation.
 Inoculating loop should be heated at first and let to cool down.
 While using inoculating loop, at first the inoculating loop should be dipped on the
sample and the sample should be transferred to broth.
 In case of tissue sample, it should be cut into small pieces and a piece of tissue should
be kept on broth.
 After performing inoculation the media should be kept at 37 °C on incubator for 24-
48 hours.

6.4 Examination of culture

We can view the growth of the bacterial colonies on culture plates with naked eyes. The
structures of the colonies also help on distinguishing the bacteria.
1. Size The study of the bacterial colonies can be done by following criteria.
2. Shape
3. Smell
4. Color
5. Consistency
6. Opacity etc
1. Size: observe the size of the colonies, colonies are measured on millimeter. The
classification of colonies can be done according to their size as follows.
 Tiny colonies= 0.1 millimeter
 Small colonies= 0.5-1 millimeter
 Medium colonies= 1-2 millimeter
 Large colonies=2-3 millimeter

The colony of streptococcus bacteria is very small.

2. Shape:
 The shapes of the colony should be observed. The general shapes of
the colonies are round, irregular, lobate, rizoid etc
 Observe the part of the colonies whether they are elevated, flat,
raised, convex etc
 The margin of the colonies should be examined whether it is filamentous
or lobate margin.
 The surface of the colonies should be examined whether it is
smooth, rough or papillae shaped.

The shape of the colony of bacillus bacteria is large and slightly dry.
 3. Smell:
The smell of the some bacteria is of unique type. Proteus bacteria have
ammonical smell, the growth of this bacteria occurs all over the plate which is
known as swarming growth.
4. Color:
The general color of the bacterial colonies is red, white, green, yellow etc. if the
bacterial colonies acquire pink color on MacConkey’s agar , we can understand
that bacteria is lactose fermenting bacteria. Staphylococcus epidermis comprises
of white color whereas staphylococcus aureus comprises of slightly yellow
colonies. Micrococcus comprises of yellow or pink colonies likewise on
MacConkey’s agar E.coli comprise of pink colony.
5. Consistency:
Examine whether the colonies are sticky or non sticky.
6. Opacity:
Bacterial colonies are transparent, translucent or opaque. On blood agar some
bacterial colonies show hemolysis. Streptococcus aureus create hemolysis on
blood agar. On blood agar Streptococcus pyogens create hemolysis around the
colonies.

6.5 Gram’s staining methods

This method was founded by the scientist named Gram on 1884 for staining the
bacteria. The bacteria are classified according to the color obtained by them while
reacting with gram’s stain. All Bacteria are classified under 2 groups

1. Gram positive bacteria

2. Gram negative bacteria

Principle:
This principle is based upon the presence of lipid. Gram positive bacteria contains low lipid
and they have contains small pores on their cell walls due to which decoloriser does not get
entrance and the bacteria take the stain of the crystal violet, as a result gram positive bacteria
obtain blue color while reacting with gram stain.

Gram negative bacteria contains high lipid and the cell wall of the gram negative bacteria
contains large pores. Because of large pores on cell wall, decoloriser (acetone) enters into the
cell wall and removes the color of crystal violet and after while the cell wall takes the color
of counter stain (safranin). Due to this reason gram negative bacterium gives red color on
gram staining.

The PH of gram positive bacteria is (2-3) and the PH of gram negative bacteria is (4-5)
6.6 Examination of milk by CMT
CMT is done on laboratory to test whether the milk sample is infected with the bacteria causing
mastitis or not.
CMT reagent can be prepared on the lab by mixing the following reagents
Name of the reagent Amount of the reagent
Sodium hydroxide 1.5 gm
Teepol 0.5 ml
Bromothylene blue 0.01 ml
Distilled water 100 ml

Test method:
 The plastic paddles containing four cups which is known as CMT
paddle is used to test the milk by this method.
 Place 3 ml of milk on each cups
 Mix 3 ml of reagent on each cup. Move the paddle on circular motion
and watch the result after 20 seconds.
 If the tested sample is Mastitis positive then the mixture of milk
and reagent become thick, viscous.
 This method can be applied on Petri Dish also.

The report of CMT on lab should be made by following the rules listed below

(-) negative= if there is no change in the mixture (0-2, 00,000 somatic cell range)

(+) trace= if the mixture contains 1-2 granules (2, 00,000-4, 00,000 somatic cell range)

(++) weak positive= if the mixture is viscous (4, 00,000-12, 00,000 somatic cell range)

(+++) distinct positive= if the mixture is highly viscous (12, 00,000-50, 00,000 somatic cell
range)

(++++) severe positive= if the somatic cell range is more than 50, 00,000

6.7 Milk culture for bacteria

Collecting sterile milk samples for bacteriological culture is the only way to definitively
diagnose infectious mastitis and identify the causative organism. Proper technique in collecting
the sterile milk sample is essential! Samples usually are collected from each quarter. Remove
dirt and water from the teats and udder; wash and dry hands; strip out the foremilk; disinfect the
teat ends with alcohol and/or teat dip (do the far teats first); remove the cap from the sterile
sample tube taking care to prevent contamination inside the tube; collect sample with tube at 45
degree angle to teat (collect the near teats first); replace cap on the tube and dip the teats.
Teat end is disinfected with an alcohol pad.

Tube is held at a 45 degree angle to prevent dirt from getting into the tube.

Milk is squirted into the tube.

Proper sample handling : Refrigerate or keep the samples on ice (less than 24 hours) or freeze.

Plating the sample : Plating the samples requires special bacteriological medium, conditions of
a laboratory and personnel with good technique in microbiology. Agitate 0.01 ml milk/quarter of
blood agar plate using a loop or pipette. For coliforms plate 0.1 ml milk/half of plates of Blood
agar and MacConkey agar plates. Incubate at 37 C for 48 hours.
 Staphylococcus aureusbacteria
on a blood agar plate.

Pathogens vs Contaminants : How do you tell if growth on the plate is a pathogen or a

contaminant? These criteria assume you are using good technique in collecting the sterile
sample and in plating the samples. Greater than or equal to 5 identical colonies in 0.01 ml milk
(pure culture) = significant. Greater than or equal to 5 identical colonies in 0.01 ml milk (mixed
culture) = significant, but questionable. Less than 5 identical colonies in 0.01 ml milk = a
contaminant.

Intrepretation : Streptococcus agalactiae and Staphylococcus aureus are usually significant

(causative agent) in any number. Bacillus is usually contaminant in any number.

Antibiotic Sensitivity Tests : are often used to determine what antibiotics may affect the
bacterium isolated from the udder. However, these take time and additional microbiological
techniques. These test use either disc diffusion or minimal inhibitory concentration assays. The
in vitro results may not reflect in vivo sensitivity of the bacterium.

Antibiotic sensitivity plate.

Reference:

1. Cha E, Bar D, Hertl JA, Tauer LW, Bennett G, et al. (2011) The cost and management
of different types of clinical mastitis in dairy cows estimated by dynamic programming.
J Dairy Sci 94: 4476–4487.
2. Jones G, Bailey T (2009) Understanding the basics of mastitis. Virginia Cooperative
Extension, Virginia Polytechnic Institute and State University.
3. Contreras GA, Rodriguez JM (2011) Mastitis: comparative etiology and epidemiology. J
Mammary Gland Biol Neoplasia 16: 339–356.
4. Makovec JA, Ruegg PL (2003) Results of milk samples submitted for microbiological
examination in Wisconsin from 1994 to 2001. J Dairy Sci 86: 3466–3472.
5. Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid
assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol
73: 5261–5267.
6. McKenna P, Hoffmann C, Minkah N, Aye PP, Lackner A, et al.. (2008) The macaque
gut microbiome in health, lentiviral infection, and chronic enterocolitis. PLoS Pathog 4.
7. Sundquist A, Bigdeli S, Jalili R, Druzin ML, Waller S, et al. (2007) Bacterial flora -typing
with targeted, chip-based Pyrosequencing. BMC Microbiol 7: 108.
8. Huse SM, Dethlefsen L, Huber JA, Mark Welch D, Relman DA, et al. (2008) Exploring
microbial diversity and taxonomy using SSU rRNA hypervariable tag sequencing. PLoS
Genet 4: e1000255.
9. NMC (1999) Laboratory Handbook on Bovine Mastitis: National Mastitis Council. 222
p.
10. Wenz JR, Garry FB, Barrington GM (2006) Comparison of disease severity scoring
systems for dairy cattle with acute coliform mastitis. J Am Vet Med Assoc 229: 259–
262.
11. Harmon RJ (1994) Physiology of Mastitis and Factors Affecting Somatic-Cell Counts. J
Dairy Sci 77: 2103–2112.