Microbiology

Microbiology
• Bacteriology
• Virology
• Mycology

The Morphology and Physiology of Bacteria

Morphology of Bacteria
Bacterial morphology describes the size, shape, arrangement and structure of the bacterial
cell.

Size
− The basic unit of measurement of bacteria is the micrometer or micron (μ).
− The micron is 1/1000 000 of a meter.
− The bacteria commonly studied in the lab measures 0.5 – 1 μ in width and 2.5 μ in length.

Shape
Bacterial cell shapes form four groups:
1. coccus (pleural cocci): spherical shape
2. Bacillus (pleural bacilli): rod shape
3. Spiral: subdivided into
i. loose spirals
ii. tight spirals
iii. comma-shaped spirals
4. Pleomorphic: coccobacilli

Cell arrangement
− Single: one cell
− Pair: e.g. diplococcus
− Tetrad: group of four
− Cluster: e.g. Staphylococcus which forms grape-like clusters
− Chains: short or long chain; e.g. Streptococcus
− Palisade: cells like Chinese letter
 Structure of Bacteria
structures common to almost bacteria
Cell wall
− The cell wall has two layers, a basal
layer and an outer layer.
− The chemical nature of the basal
layer is the same in Gram positive
and Gram negative bacteria but that
of the outer layer is different.
− The outer layer is thick in Gram
positive bacteria (90% peptidoglycan)
and thin in Gram negative bacteria
(10% peptidoglycan).
− The cell wall maintains shape and
size.
Cell membrane (cytoplasmic membrane)
− The cell membrane is the innermost
layer separating the cytoplasm from
the cell wall.
− It is similar in structure to the
mammalian cell membrane.
− It is semipermeable membrane which
controls the passage of materials in − More than one pilus may be present on the cell.
and out.
− Contain mesosome that increase the − Not involved in motility and can be found in motile or nonmotile
surface area for secretion and cell
division.
Cytoplasm
− Is a complex fluid mass containing
ribosomes and granular inclusions,
which are mainly storage granules
(source of energy).
− The cytoplasm also contains the
nuclear area which contains DNA.
structures characteristic of certain bacteria
Capsule
− Is a jelly-like material forming the outermost layer of many bacteria.
− The capsule functions are:
1. To protect bacteria against phagocytosis.
2. To produce infectivity (virulence or pathogenicity).
3. To prevent nutrient loss and dehydration.
4. To provide antigenicity and causes production of specific
antibodies useful in diagnostic.
Flagella (singular flagellum)
− Flagella emerge from the outer layer of the cell wall.
− They are composed of protein.
− They are organs of motility.
− There are 4 types of flagella:
A. monotrichous: has a single flagellum at one end.
B. lophotrichous: has many flagella at one end.
C. amphitrichous: has a flagellum at both ends.
D. peritrichous: has flagella all around the cell.
Pili (singular pilus)
− They emerge from the basal layer of the cell wall.
− They are mainly found in Gram negative bacteria.
bacteria.
− They allow the bacteria to attach and adhere with the host.
− They allow for exchange DNA between bacteria.
− They determine the pathogenicity; e.g. E. coli must have pili to cause
disease.
Endospores
− Spores can be central, located in the center of the cell.
− Spores can be subterminal, located near the end of the cell.
− Spores can be terminal, located at the end of the cell.
Nutrition and Growth of Bacteria
• The essential elements needed for growth are
o Carbohydrates
o Proteins
o Lipids
o Water
o inorganic salts
o trace elements
• Other factor affecting growth, are temperature, oxygen, carbon dioxide and pH.
Temperature
According to temperature requirement bacteria can be grouped into:
a. psychophiles: these are cold-loving bacteria; they grow at 0 – -30°C.
b. mesophiles: these bacteria grow at 20 – 40°C. Most pathogenic bacteria
belong to this group, but the optimum temperature for most animal
pathogens is 35 – 37°C.
c. thermophiles: these grow best at 40 – 80°C.

Gases
According to gas requirement, bacteria can be grouped into:
a. aerobic: grow in presence of oxygen.
b. anaerobic: grow only in absence of oxygen.
c. facultative anaerobic: grow in absence or presence of oxygen but must
obtain oxygen-containing compound such as inorganic sulfates.
d. microaerophilic: require oxygen less than what in air.
e. capnophilic: require 3 – 10 % carbon dioxide.

pH
The optimum pH for most pathogenic bacteria is 6.5 – 7.5.

Osmotic pressure
Osmotic balance maintains the cell integrity so that it grows normally.

• Bacteria can be divided into 2 groups based on their metabolism:

1. photosynthetic bacteria: these use light as a source of energy.
2. chemosynthetic bacteria: these oxidize chemical compounds to get their energy.
There are 2 types of chemosynthetic bacteria:
i. autotrophic bacteria: get their energy from an inorganic source. They are not
of veterinary importance.
ii. heterotrophic bacteria: get their energy from an organic source. They are of
veterinary importance.
 Reproduction of
bacteria
asexual
)binary fission)
The cell increases in size, elongate
and divide into two after
redistribution of cellular material.
A newly formed bacterium
requires 15 – 30 minutes to reach
adult size and divide.
Sexual
There is no meiosis, formation of gametes and zygote
Transformation
DNA transfer from virulent
capsulated bacterium into
avirulent non-capsulated
bacterium. This produce virulent
capsulated bacterium.
Transduction
DNA transfer from a donor bacterial
cell into a recipient bacterial cell
with the help of a bacteriophage.
Conjugation
DNA transfer from a male bacterial cell
(donor) into a female bacterial (recipient).
The donor cells have a fertility factor (F
factor) as a component of its circular DNA
while the recipient cell does not have this
factor. This produce F+ bacterial cells
Bacterial growth Bacterial growth curve
Bacterial culture in under suitable conditions proceeds
through at least 5 phases:
1. Lag phase: the phase of adjustment of bacteria. No
growth takes place during this phase.
2. Logarithmic phase: during this phase the number of
bacteria doubles. Cells in this phase are young, uniform
and viable.
3. Stationary phase: growth rate and death rate are equal
(plateau).
4. Death or decline phase: during this phase the growth
rate declines because the number of dying bacteria more
than bacteria produced.
5. Dormant phase: during this phase, few numbers of
bacteria survive.

Colony morphology
Determining the morphology of a single colony
1. Measure the colony size in millimeters.
2. Describe the pigmentation.
3. Describe the form, elevation, and margin.
 Types of culture media

Based on their consistency Based on the constituents/ingredients Based on Oxygen requirement

1. solid medium - 2% agar 1. simple medium (basal medium) 1. Aerobic media
▪ Colony morphology, ▪ E.g. nutrient broth (NB), nutrient agar (NA) 2. Anaerobic media
pigmentation, hemolysis ▪ NB consists of peptone, meat extract, NaCl
can be recognized. ▪ NA = NB + 2% agar
2. semi solid medium - 0.5% agar 2. complex medium
▪ E.g. Motility medium. ▪ Added ingredients to provide special nutrients.
3. liquid medium (Broth) - no agar 3. synthetic or defined medium
▪ Prepared from pure chemical substances.
▪ E.g. Nutrient broth
4. special media
– Enriched media
▪ Substances like blood, egg, serum are added to
the solid simple medium.
▪ Used to meet the requirements of fastidious
bacteria which need extra nutrients to grow.
▪ E.g. Blood agar, Chocolate agar

– Enrichment media
▪ Added inhibitory substances to liquid media to
kill the unwanted bacteria and isolate particular
organism.
– Selective media
▪ Added inhibitory substance to a solid media.
– Indicator media
▪ Added indicator to indicate certain reaction
where the color of indicator changes when
reaction happen.
▪ E.g. urease medium
– Differential media
▪ Added substances to differentiate between two
reaction.
▪ Must contain indicator and inhibitory substance.
▪ Differentiate between lactose fermenters &
non-lactose fermenters.
− Lactose fermenters – Pink colonies
− Non-lactose ermenters – colourless colonies
– Transport media
▪ Media used for transporting the samples to keep
microbes alive.
▪ Usually it is semisolid.
Gram staining
The Gram Stain Procedure
1. Prepare a Smear: to kill bacteria and fix them onto the slide
– Add bacteria in a drop of water on a microscope slide.
– Heat the slide.
2. Add Crystal Violet: violet colored,
stains all microorganisms.
– Rinse with water to remove excess
stain.
3. Add Iodine: act as mordant to complex
violet color.
– Rinse with water to remove excess
Iodine.
4. Add Decolorizer: dissolves
lipopolysaccharide layer of Gram-
negative organisms.
– Drip Alcohol (80% Methanol +20%
Acetone).
– Rinse with water to remove excess alcohol.
5. Add Safranin: Stains the wall of Gram-negative organisms in red color.
– Flood the slide with Safranin solution.
6. Rinse, Dry and Observe
– Rinse with water to remove excess stain Blot dry.
– Observe under Oil Immersion.

Potassium Hydroxide (KOH) Test

• Used when bacteria stained both gram positive and negative, which is called gram
variable.
• Gram variable causes:
1. Thick smear
2. Excessive heat fixation
3. Excessive decolorization
4. Old culture
5. Poor quality of stain
• Procedure of Potassium Hydroxide Test:
1. Place on a slide 3% of KOH solution.
2. Take generous quantity of bacterial growth with loop from the culture and stir it
into the KOH drop.
3. After a maximum of 2minutes of stirring, gram negative bacteria become mucoid
and produce a sticky strand when the drop is lifted with the loop. If the bacteria
are gram positive the mixture stay homogeneous and doesn’t form strand on
lifting.
 Enumeration
(counting)

Standard plate count (Viable count) Spectrophotometer

▪ A set of serial dilutions is made. ▪ Depends on turbidity.

▪ Find plate with 30-300 colonies. ▪ Live and dead count.
▪ Colony-forming units, CFUs.
▪ C (CFUs) = N (colony count from the plates) = CFUs/ml
D (dilution factor) x S (volume plated)
▪ Dilution factor = amount of specimen transferred divided
by the total volume after.

Average = 225

C= 225 = 2.25x10-9 CFUs/ml

-6 -1
10 x 10
 Gram-positive Bacteria

G+ cocci
.
G+ coccobacilli G+ bacilli
Catalase test

Micrococcaceae Streptococcaceae
▪ Aerobic or facultative anaerobic bacteria. ▪ Anaerobic or Facultative anaerobic bacteria.
▪ Catalase positive. ▪ Catalase negative.
▪ Produce catalase enzyme. ▪ Don’t produce catalase enzyme.
▪ Catalase converts hydrogen peroxide (H2O2) ▪ Arrangement
to water (H2O) and oxygen gas (O2) for protect − diplo, tetrad and chain
the bacteria from breaking down. ▪ Classified on the basis of their antigen.
▪ H2O2 is bactericidal. ▪ Lancefield grouping
▪ Place a colony from blood agar to microscope
.
− A, B, C, D, F, G
slide and add a drop of H2O2. If the colony is ▪ Usually non-motile.
catalase positive, gas bubbles will produce. ▪ Fastidious, requiring enriched media.
▪ Commensals on mucous membranes.
Staphylococci ▪ Cause pyogenic infections.
Micrococci
▪ hemolysis
▪ facultative anaerobic. − produce hemolysin.enzyme
▪ Contains both pathogenic and non-pathogenic organisms.
▪ Staphylococcus major species: (β-hemolysin) cause (α-hemolysin) cause (γ-hemolysin)
− Aureus: Pathogenic, cause Mastitis, yellow. complete destruction of partial destruction, No destruction,
− Epidermidis: Non-pathogenic. RBC, Blood agar will Blood agar will Blood agar not
− Intermedius: Photogenic, cause Mastitis and become yellow become green change
Pyoderma in dog, white. ▪ CAMP test (Christie-Atkins, Munch-Petersen)
− Pseudintermedius: Pathogenic, causes Pyoderma and − Used to differentiate between streptococcus
Otitis externa in dog. groups A, B, C, D, F, G
− Hyicus: Pathogenic, cause Mastitis and Polyarthritis.
▪ Coagulase test: used to determine pathogenicity of CAMP test
Staphylococcus only.
− Need plasma (rabbit plasma) to provide fibrinogen.
CAMP + CAMP -
− convert fibrinogen to fibrin.
− Bacterial cell covered with fibrin to hide from the ▪ Group B
immune system. ▪ Staphylococcus aureus produce β-hemolysin
− Coagulase positive is pathogenic.
.

enzyme which reacts with CAMP factor produced by

− Coagulase negative is non-pathogenic. both b-hemolytic and nonhemolytic Streptococcus
then a yellow arrow appears that indicates a
slide coagulase test tube coagulase test positive CAMP reaction (synergistic interaction).

▪ Bound coagulase ▪ Free coagulase

▪ Bacteria produce clumping
factor to help coagulase in
Rabbit plasma
binding with fibrinogen.
incubate at 37°C
1. Streak Staphylococcus aureus onto a sheep blood agar
for 1.5 hours plate.
2. Streak a straight-line inoculum of group
B Streptococcus but not touching, the S. aureus streak.
3. The plates are incubated at 35°C for 24 hours.
Blood Agar
• Blood Agar is a general-purpose medium.
• The quadrant streak technique tips:
o Flame the loop until it is red hot and then allow it to cool.
o Take a small amount of bacterial growth with the sterile loop.
o Immediately streak the loop very gently on a quarter of the
plate.
o Flame the loop again and allow it to cool. Going back to the
edge of area 1, extend the streaks into the second quarter of
the plate (area 2).
o Flame the loop again and allow it to cool. Going back to the
area 2, extend the streaks into the third quarter of the plate
(area 3). Subculture blood agar
o Flame the loop again and allow it to cool. Going back to the area 3,
extend the streaks into the center fourth of the plate (area 4).
• Quadrant streaking should be used to get well-isolated colonies.
• Usually we get a few isolated colonies in the third or fourth quadrant.
• Flame the loop after you streak each quadrant to dilute the bacteria.

MacConkey agar
• MacConkey agar is a selective and differential media.
• MacConkey agar is used for the isolation of gram-negative 1. Crystal violet
enteric bacteria and the differentiation of lactose fermenting 2. Bile salt
3. lactose
from lactose non-fermenting gram-negative bacteria.
• Crystal violet and bile salts prevent the growth of G+ bacteria.
• Growth of lactose fermenter appears pink on the agar and
MacConkey agar
growth of non-lactose fermenter appears colorless and translucent.

Mannitol salt agar (MSA)

• Use only to grow Staphylococci.
• MAS contains:
o Salt (7.5% NaCl) as selective ingredient, select only for staphylococci and
no other bacteria can grow because the high concentration of the salt 1. 7.5% NaCl
2. Mannitol
affects the osmotic pressure.
3. Phenol red
o Mannitol as differential ingredient, mannitol fermenter produces acidic
product that reduce pH at level below 6.9 then the medium become
yellow color, while the medium in mannitol non-fermenter remain red. MSA
o phenol red as a pH indicator (pH= 7.8) in the medium.
• Mannitol fermenter is pathogenic.
• Mannitol non-fermenter is non-pathogenic.
DNase Test
▪ White blood cells called neutrophils have a two-pronged defense against
bacteria:
− They can engulf and destroy them.
− They can release neutrophil extracellular traps (NETs), which made up of
DNA and toxic compounds that can catch and kill pathogenic bacteria that
produce DNase enzyme to destroy the DNA of NETs.
▪ DNA Hydrolysis test or (DNase) test is used to determine the ability of an
organism to hydrolyze DNA and utilize it as a source of carbon and energy for
growth.
▪ DNase agar is a differential medium used to test the ability of an organism to
produce DNase enzyme.
▪ DNase indicator:
− Methyl green
• If it is pathogenic the DNA will hydrolysis. So, the clear colorless halo
appears around the test organism.
− Toluidine blue
• If it is pathogenic the DNA will hydrolysis. So, the medium turns to pink
color around test organism.
− Hydrochloric acid (HCL) only when DNase agar without indicator
• If it is pathogenic it will produce a clear zone around the test organism. If it
is nonpathogenic the medium will become opaque.
▪ There are two types of inoculation that can be done.
− Spot Inoculation
− Band or line streak inoculation
Use a heavy inoculum and draw a line 3-4 cm long from the rim to the center of the
DNase test agar plate.
 Mastitis
• Inflammation of one or more quarters of the udder.
• Causes by aureus staphylococcus.
• Mastitis due to E.coli became a serious problem due to
growing size of dairy herds kept in close confinement
during winter.

Subclinical Mastitis Clinical Mastitis

− %90- 95 ~of all mastitis cases − %5 - 10 ~of all mastitis cases
− Udder appears normal − Inflamed udder
− Milk appears normal − Clumps and clots in milk
− Elevated somatic cell count SCC (score 3-5) − Acute type
− Lowered milk output)%10 ~( ▪ major type of clinical mastitis
− Longer duration ▪ bad milk
▪ loss of appetite
▪ depression
▪ attention needed
− Chronic type
▪ bad milk
▪ cow appears healthy
What causes mastitis ?

Bacteria ( ~ 70%) Yeasts and molds ( ~ 2%) Unknown ( ~ 28%)

− physical
Where do these organisms come from? ▪ trauma
• Infected udder ▪ weather extremes
• Environment
− bedding
− soil
− water
− manure
• Replacement animals
How is mastitis diagnosed?
• Physical examination
o Signs of inflammation
o Empty udder
o Differences in firmness
o Unbalanced quarters
• Cow side tests
o California Mastitis test (CMT)
o Qualitative not quantitive.
o CMT steps
1. Disinfected the udder.
2. Squeezed first catch milk (very contaminate).
3. 2nd catch milk 5-10 ml.
4. Put the milk on the paddle.
5. Add reagent into the milk and shake the paddle.
Shaking to prevent the attachment between somatic cells and fat of milk.
6. Gel formation = Mastitis / No gel formation = No mastitis
Gel formation because of the reaction between DNA of somatic cells and
reagent.
• Culture analysis
o The most reliable and accurate method

• Analytical profile index (API) staph test

o Differentiate among staphylococcus species.
o API consist of a strip containing dehydrated
test substrates in individual microtubes.
o Done by one colony of bacteria.
o Depend on gram staining, catalase test and
subculture.
o Incubate one day at 37°C.
o After incubation, positive test results are scored as a seven-digit number.
 Reagent control
Kit card
Latex agglutination test for staphylococcus aureus
• Cell wall of the staphylococcus aureus contains antigen called protein A. A B +
• Latex kit is used to detect the presence of protein A.
• Latex particles coated with IgG antibodies and fibrinogen which reacts A B C D G
with protein A of staphylococcus aureus and produce agglutination if it is pathogenic.

Treatment of staphylococcus
• All kind of pathogenic staphylococcus have resistance to penicillin because the
staphylococcus produces β-lactamase enzyme which destroy β-lactam ring in the
penicillin so penicillin cannot work.

Antibiotic sensitivity test (AST)

• Measure the ability of an antibiotic or other antimicrobial agent to inhibit the in
vitro microbial growth.
• The results of this test indicate which antimicrobial to use in treatment.
• Muller Hinton agar
− Better diffusion allows diffuse of antibiotic.

Inhibition zone
24h (no growth of bacteria)
37°C

− Depend on diameter
1. Sensitive (S), when presence of inhibition zone which means that
antibiotic inhibit growth of bacteria.
2. Intermediate (I)
3. Resistant (R), when absence of inhibition zone which means growth of
bacteria because antibiotic not able to inhibit bacterial growth.

Streptococcus
• Sometimes It has capsule.
• It’s cell wall contain C-component which consider as antigen of streptococcus.
• C-component different according to the lancefield group and each one has certain
antibody.
• Extract C-component to know the group of bacteria by using reagent 1 and reagent 2
which contain a chemical substance able to extract the antigens at room temperature.

kit
Streptococci of Veterinary Importance
• S. pyogenes
− Group A, CAMP –
− Pyogenic infection
− Human pathogen
− Dog and cat are reservoir
− Cause bovine mastitis (rare) and respiratory infection
• S. agalactiae
− Group B, CAMP +
− Pyogenic infection
− Cause mastitis
• S. dysgalactiae
− CAMP –
− Pyogenic infection
− Cause bovine mastitis and polyarthritis (lamb)
• S. equi
− Pyogenic infection
− In horse
− Cause strangles disease (very common), mastitis and genital infection
• S. zooepidemicus
− Pyogenic infection
− Effect horse, cattle and swine
− Very pathogenic for poultry
− Cause cervicitis, metritis, septic arthritis and abortion in mare
• S. enterococcus faecalis
− Opportunistic bacteria
− γ-hemolytic
− Commensal in cattle intestine
− Highly pathogenic for poultry
− common inhabitant of the intestinal tract of born human and animals
• S. uberis
− Cause bovine mastitis
− Recovered from the tonsils
− Vagina of cattle
• S. pneumoniae
− In upper respiratory tract of animals

Treatment
In general streptococci are sensitive to penicillin.
Strangles disease
• Strangles, which also is known as horse distemper.
• Highly contagious disease of horses.
• caused by the bacteria Streptococcus equi, which infects the upper respiratory tract of
equine species (horses, mules, zebras).
• Horses become infected with strangles after inhaling or ingesting the bacteria.
• Strangles can occur in any age horse, but are infected more frequently horses between
1 and 5 years old.
• Discharge from the nose or abscesses, carries large concentrations of the bacteria.
• Four Stages of Disease:
1. Early Stage of Infection
Initial symptoms may include depression, failure to eat or drink, clear nasal discharge,
high fever and swelling of the lymph nodes under the jowl or in the throatlatch area.
2. Lymph Node Abscesses
Abscesses may develop in the swollen lymph nodes 7 to 10 days after initial signs
are observed. As the abscesses mature, they will rupture and drain thick cheesy
material.
3. Exposed But No Symptoms
The first clinical signs usually develop within 2 to 6 days after exposure, although
having the incubation period last 14 days is not uncommon.
4. Complications
A condition known as "bastard strangles" can occur in up to 20% of cases. This
condition results when abscesses form throughout the body, with the most common
locations being the lungs, liver, spleen, kidney and brain.
• Treatment
Treatment is highly dependent on veterinarian preference and the stage of the disease.
Streptococcus equi is very sensitive to a variety of anti-microbials.
• Prevention/Control
Isolating affected horses immediately is essential for the prevention and control of
strangles. Any equipment, including all brushes, buckets and tack, that has come in
contact with an affected horse should be disinfected thoroughly.
• Vaccination
The unique strangles vaccine can be used in horses from just 4 months of age and is
administered through injection into the upper lip of the horse.
 Corynebacterium species
General characteristics:
• Gram-positive.
• pleomorphic shape (coccobacilli).
• Non-motile.
• Non spore forming.
• Fastidious, requiring enriched media to grow.
• Facultative anaerobic.
• Catalase-positive.
• Urease positive.
• Oxidase-negative.
• Non-lactose fermenter.
• Cause pyogenic infections.

Corynebacterium species

C. bovis C. pseudotuberculosis C. renale

− Cause subclinical Mastitis. − Infect sheep and goat. − Cause Bovine pyelonephritis.
− Cause Caseous Lymphadenitis. − Clinical sign
− Characterised by abscessation and • Fever
enlargement of lymph nodes. • Decreased milk
− Cell wall has lipid and it is production
capsulated. • Kicking the abdomen
− Diagnosis • Dysuria
1. Direct gram staining • Bloody urine
2. Elisa for exotoxins − Diagnosis
3. API 1. Unilateral thickening of
4. Urease test ureters and enlarged
5. Nitrate test kidneys by rectal
palpation.
2. Culture of C. renale from
urine.
3. Direct gram staining
4. Elisa for exotoxins
5. API
6. Urease test
7. Nitrate test
 Urease test
− The urease test is used to determine the ability of an organism to split urea into two
units of ammonia with CO2, through the production of the enzyme Urease.
− CH4N2O + H2O Urease CO2 + 2NH3
(Urea) enzyme
− The pH indicator phenol red which under acid conditions (pH 6.8) is yellow.
− Ammonia rise pH over (pH 8.4) so the urea agar turns from yellow to pink.
− Urea agar is semisolid agar contain: 20g urea, 1g gelatin peptone, 5g NaCl, 1g dextrose,
phenol red indicator and monopotassium phosphate.
− It done in the lab by streak the slant of urea agar with test organism then incubate the
tube at 35-37°C for 24 hours. Pink color indicates positive result and yellow color
indicates negative result.

Nitrate reduction test

− A test to differentiate between bacteria based on their ability or inability to reduce
nitrate (NO3) to nitrite (NO2-) using anaerobic respiration.
− Addition sulfanilic acid and α-naphthylamine, turned broth to red due to their reaction
with nitrite, indicating nitrate reduction. If there is no color change, nitrite is absent.
− Some of bacteria have enzymes to further reduce the nitrite to either the ammonium
ion or nitrogen gas.
NO3 Nitrate reductase NO2- Further reduction NH4 or N2
Nitrate enzyme Nitrite Ammonium Nitrogen
ion gas

Inoculate the
colony Incubation Add drops of Force
NO3 37C NO2 Sulfanilic acid and reduction
α-naphthylamine
Nitrate Broth If no red color appears, If turn to red color, indicates no nitrate reduction.
add zinc powder + HCL as If no red color, indicates nitrate reduction.
force for nitrate reduction
Endospore forming
− Endospores are resistant against:
• Drought
• Low nutrient conditions
• Radiation
• High temperatures
• Various chemical disinfectants
− Endospore staining is done by using old culture.
− Stain Procedure:
1. Make a smear and heat fix the organism.
2. Cover the smear with square of blotting paper and flood it with Malachite Green
stain solution for 10 minutes.
3. Wash the slide with water.
4. Counter stain with Safranin for 30 second, then wash the slide with water and dry it.
5. Examine the slide under oil immersion lens.

Spore forming Bacilli
Bacillus
• Catalase +
• Aerobic
• Bacillus anthracis
o Gram +
o Facultative anaerobic
o spore-forming
o has capsule composed of
protein, helps to evade the
immune system through
preventing phagocytosis by
macrophages.
o produces and releases an
exotoxin composed of three
proteins:
1. edema factor
2. protective antigen
3. lethal factor
o Each protein by itself is nontoxic,
but together they produce the
systemic effects of anthrax.
o Anthrax three forms:
cutaneous anthrax
• The most common form.
• The exotoxin causes a painless
round black area on the skin 2 to
5 days after exposure, the result
of localized tissue necrosis.
• Treatment by penicillin.
G+ Bacilli
Non spore forming Bacilli
Clostridia
• Catalase -
• Oxidase -
• Anaerobic
• Require enriched media for
growth.
• Produce endospores.
• The size, shape and location of
endospores can be used for
species differentiation.
Respiratory anthrax Gastrointestinal anthrax
• Inhalational anthrax occurs when the • A rare form.
spores are inhaled. • caused by ingestion of spores
• The bacilli mature and replicate in the from improperly cooked,
lungs, where the exotoxin is released. contaminated meat.
• Symptoms, which include fever, cough, • Bacilli is able to mature and
malaise, fatigue, and body aches. replicate within the intestine,
• generally, develop within 7 days of causing a necrotic lesion.
exposure. • Symptoms include: vomiting,
• Respiratory distress and death quickly abdominal pain, and bloody
follow in 95% to 100% of untreated diarrhea.
cases. • Mortality rates are high (25% to
• Laboratory Diagnosis: 60%) if untreated.
o cut the small ear vein and several • Treatment by penicillin.
smears are made directly on slides.
o or the blood is absorbed onto a
swab.
 Clostridia

neurotoxic clostridia histotoxic clostridia enteropathogenic

• affect neuromuscular function without • produce localized lesions in tissues • produce inflammatory
tissue damage. such as muscle and liver. lesions in the gastrointestinal
• Two types • may cause toxemia. tract.

Clostridium tetani Clostridium botulinum

− Cause tetanus disease. − Cause botulism toxin.

− Has terminal spore with ‘drumstick’ appearance.
− Has a swarming growth (not isolated colony) and
is hemolytic on blood agar due to the production
of tetanolysin toxin.
− Has flagellar antigens used to differentiate
between the 10 serotypes of C. tetani.
− The neurotoxin tetanospasmin is responsible for
the clinical signs of tetanus and causes paralysis.
− Infection occurs when endospores are introduced
into traumatized tissue from soil or feces.
− Common sites of infection
1. Deep penetrating wounds in the horse.
2. Castration, docking and dehorning wounds in
sheep.
3. Abrasions associated with dystocia in cows
and ewes.
− The presence of necrotic tissue, foreign bodies
and contaminating facultative anaerobes in
wounds may create the anaerobic conditions in
which C. tetani spores can germinate.
− Tetanus toxin binds to receptors on motor
neuron terminals and is transported to the nerve
cell body, so it blocks the messages from brain to
muscle, by retrograde intra-axonal flow which
cause paralysis.
• Diagnosis of Clostridium
− Mouse inoculation by taking the serum from infected
animal and inoculates in the mouse.
− Toxin neutralization tests in mice, by Elisa test and
observe change in color by plasma.
− Culture for detect Colonial morphology.
− Biochemical test (API, PCR).
• Treatment of Clostridium
− If available, polyvalent antiserum is effective in
neutralizing unbound toxin at beginning of the disease.
− Usually acquired by ingestion of pre-formed
toxin.
− Produces oval, subterminal endospores.
− 7 types of Clostridium botulinum are recognized,
based on the antigenic properties of the toxins
(A, B, C, D, E, F, G) which they produce.
− The C-beta and D toxins cause botulism in cattle,
sheep, equine, and sporadically in other species.
− The disease is presented in two ways:
▪ Emergence of isolated cases or small
outbreaks usually due to the ingestion of
contaminated feed.
▪ Occurrence of epizootic outbreaks Related to
abnormal ecological factors, such as
Phosphorus deficiency in pastures which
causes animals to ingest bones to
supplement this mineral.
− When the animals die aerobic bacteria
present in the digestive tract, creating
anaerobic condition for spore
development. These spores produce
toxin and this toxin contaminates porous
bones, tendons, and ligaments causing
the carcass to be the main link in the
epizootic chain.
− The clinical signs of botulism, which develop 3 to
17 days after ingestion of toxin, are similar in all
species.
▪ Dilated pupils, dry mucous membranes,
decreased salivation, tongue flaccidity and
dysphagia in farm animals.
▪ Paralysis of respiratory muscles leads to
abdominal breathing and death.
 Gram-negative Bacteria

Enterobacteriaceae Non-Enterobacteriaceae
(Enterics) (Non-Enterics)
• facultative anaerobic rods. • oxidase positive (cytochrome oxidase).
• Motile or non-motile.
• Catalase positive.
• oxidase negative (no cytochrome oxidase).

Opportunistic diseases gastrointestinal diseases

• Septicemia.
• Pneumonia.
E.coli Salmonella Shigella Yersinia entercolitica
• Meningitis.
•
• urinary tract infections.
•
Motile by means of peritrichous flagella.
lactose positive
lactose negative
• Indole positive
• Grows well on nutrient agar at 37°C.
• Colonies may be smooth or rough.
• Usually O157:H7
• Serotypes Has 3 surface antigens
o O antigen (lipopolysaccharide)
o H antigen (flagellar)
o K antigen (capsular)
• Neonatal infections in calves, swine, sheep and puppies.

Colibacillosis Calf scours

• Infect calves, piglets, lambs, pigs. • occurs in animals less than 2 weeks old.
• occurs in 2 forms • Caused by E. coli K 99
• predisposing factors include:
o Lack of colostrum intake.
septic Enteric o Cold wet conditions.
• more common in 2-3 • similar to calf o Over-crowding.
weeks old lambs. scours. • Symptoms:
• show septicemia and o fever
sudden collapse. o diarrhea (feces are profuse, liquid
yellow or grey)
o dehydration
o may results in death
Laboratory diagnosis
• Can be isolated from the feces.
• Direct gram staining.
• Plate the fecal sample on blood and MacConkey agar.
• Inoculate TSI, citrate, indole, methyl red for biochemical test.
o lactose fermenters give yellow color on TSI slant, urease & citrate
negative and Indole positive.
Treatment:
1. Antibiotic (ampicillin, chloramphenicol).
2. fluid therapy.
Control
• Good colostrum intake during the first few hours of life.
• Proper housing and good hygiene.
• Vaccinating susceptible animals.
 gastrointestinal diseases

E. coli Salmonella Shigella Yersinia entercolitica

• gram negative
• Motile with the exception of Salmonella Gallinarum and
Salmonella Pullorum.
• Facultative aerobes
• Catalase positive
• Lactose negative
• Oxidase negative
• Indole negative
• grow at an optimum of 37°C.
• Optimum pH for growth of Salmonella is 6.5-7.5; may grow
at a pH range of 4.5-9.0.
• Salmonella infect all types of domestic animals.
• One of the most prevalent agents causing food-borne
disease- salmonellosis.
• S. enterica is divided into seven subspecies.
• Examples: Typhimurium, Enteritidis, Choleraesuis, Dublin,
Gallinarum and Pullorum.
• Poultry products remain the main reservoir of salmonella.
• Disease depends on species and age of the host-more
severe in newborns, infants and the elderly.
• Host specific
o typhi, paratyphi, sendai cause disease only in humans.
o choleraesuis in pigs can also infect humans.
o S. Dublin in cattle.
o S. Gallinarum and S. Pullorum in poultry.
o typhimurium and enteritidis are the major serovars
that cause disease in humans, cattle, poultry, sheep,
pigs, horses and wild rodents.
• A complication of infection is the establishment of a carrier
state which lasts for 5-6 months.
• 2 forms of the infection in animals

Enteric Septic
• Young animals are the
acute enteritis Chronic enteritis most susceptible.
• Characteristics:
• is more common in older animals. • more common also in older animals.
o No diarrhea
• Characteristics: • Characteristics: o High fever
o Severe watery diarrhea o Persistent diarrhea
o Increased respiration
o High fever o Severe emaciation and pulse
o Rapid pulse and respiratory rates o Intermittent fever o Neurological signs
o Feces have putrid odor and may o The feces may be blood tinged o Mortality is very high
contain mucus. and contain mucus.
approaching 100 %
o Food poisoning is seen in
humans but not animals.
Salmonella Invasion
• Salmonella passes through the stomach and survives the acid pH by eliciting an acid
tolerance response then colonizes in the ileum through entr from the M-cell and binds
to tissues of ileum by means of fimbriae.
Salmonella Diagnosis
• Colorless on Mackonkey agar.
• Bright pink colonies on brilliant green agar.
• Indole negative.
• Urease negative.
• Citrate positive.
• Serological tests such as ELISA and agglutination test is the most useful in diagnosis of
Salmonella.
Salmonella treatment
• Antibiotic therapy should be based on susceptibility testing.
• Antimicrobial therapy should be used with care to treat enteric salmonellosis because it
may disturb the normal intestinal flora.
• Fluid therapy is required to control dehydration and shock.
Oxidase test
• The oxidase test is used to determine the presence of cytochrome oxidase enzyme by
using a reagent, tetra-methyl- p-phenylenediamine dihydrochloride, as
an artificial electron donor for cytochrome.
• It done in the lab by streak the bacteria in paper soaked with the reagent,
If the bacteria streaked in paper are Non-Enterics the cytochrome oxidase
enzyme will react with the reagent, and the color of paper will change
from colorless to dark purple within 10-30 seconds.

Citrate test
• Tests for the ability of bacteria to convert citrate into acetic acid
and oxaloacetic acid. The oxaloacetic acid then hydrolyzed into
pyruvic acid and CO2.
• In this media, citrate is the only carbon source available for the
bacteria. If bacteria cannot use citrate, it will not grow.
• If bacteria can use citrate, it will grow and the media will turn
blue as a result of increasing the pH.

Indole test
• The indole test screens for the ability of an organism to degrade the amino acid
tryptophan and produce indole.
• It is used as part of the IMViC procedures.
o IMViC (Indole test – Methyl red test – Voges-Proskauer test – Citrate test)
• Group of tests to distinguish among members of the family Enterobacteriaceae.
• Tryptophan deamination and hydrolysis by Tryptophanze system
o tryptophan + water = indole + pyruvic acid + ammonia
• Detection of indole, a by-product of tryptophan metabolism, relies upon the chemical
reaction between indole and p-dimethylaminobenzaldehyde (DMAB) under acidic
conditions to produce the red ringe.
• PROTOCOL
o Inoculate the tube of tryptone broth with a small amount of a pure culture.
o Incubate at 35°C (+/- 2°C) for 24 to 48 hours.
o To test for indole production, add 5 drops of Kovács reagent (contain p-
dimethylaminobenzaldehyde) directly to the tube.
o A positive indole test is indicated by the formation of a
pink to red color ("cherry-red ring") in the reagent
layer on top of the medium within seconds of adding
the reagent.
o A negative indole test has not broken down the
tryptophan in the medium and thus no color change
occurs upon addition of the Kovács reagent. The
reagent appears as a thin yellow layer on top of the
culture medium.
Triple Sugar Iron Agar (TSI)
• A semisolid tubed differential medium used to determine sugar fermentation and H2S
production.
• TSI is most frequently used in the identification of the Enterobacteriaceae, although it
is useful for other gram-negative bacteria.
• Bacteria can ferment sugars aerobically (with oxygen) or fermentatively (without
oxygen) .
• TSI contains three sugars: glucose (0.1%), sucrose (1%), and lactose (1%).
• The medium also contains peptones which are the sources of nitrogen, beef extract,
yeast extract, vitamins and minerals, Phenol red as the pH indicator, and agar is used
to solidify the medium.
• During preparation, tubes containing molten agar are angled. The slant of the medium is
aerobic, while the deep (or butt) is anaerobic.
• When any of the sugars are fermented, the drop in pH will cause the medium to change
from reddish-orange (the original color) to yellow.
• A deep red color indicates alkalization of the peptones.
• The result acid over acid (A/A) indicate the fermentation of glucose, sucrose and/or
lactose. The bacteria quickly fermented the glucose, producing an acid slant and an acid
butt in a few hours. After further incubation (18 hours) the glucose was consumed, and
then the bacteria fermented sucrose and/or lactose, maintaining an acid slant.
• The result is alkaline over acid (K/A) because the bacteria fermented glucose and could
not ferment sucrose or lactose, then peptones (amino acids) utilized aerobically on the
slant and released ammonia (NH3) which increased the pH and turn slant from yellow
to red.
• The result alkaline over acid (K/A) with black precipitate. The black precipitate
indicates the production of hydrogen sulfide (H2S) from reducing of sodium thiosulfate.
The hydrogen sulfide will react with ferric ions in the medium to produce iron sulfide, a
black insoluble precipitate.
• The result alkaline over alkaline (K/K) indicate that the peptones utilized both
aerobically and anaerobically, the slant and butt will be red.
• If peptones only utilized aerobically, the slant will be red and the butt will be not
change (K/NC).
• Gas production (CO2 and O2) is detected by splitting of the agar. In some cases, so
much gas is produced that the agar is pushed to the top of the tube.
• PROTOCOL
o Use a straight inoculating needle to pick up an isolated colony.
o Inoculate the TSI slant by first stabbing the butt down to the
bottom, withdraw the needle, and then streak the surface of
the slant. Use a loosely closure to allow access of air.
o Read results after incubation at 37°C for 18 to 24 h. Three
kinds of data may be obtained from the reactions.