Microbiology
Clinical Pathology
Microbiology
• The study of microscopic organisms
• Clinical microbiology is the identification of
these organisms that cause clinical illness
Laboratory Safety
• Most microorganisms are potentially
pathogenic
• DO NOT EAT OR DRINK IN LAB!
• Clean area with disinfectant at
beginning and end of the work period
• Avoid putting any object in mouth
(pencil, fingers).
• Tape shut all containers of cultures
before disposal.
• Flame wire loops immediately after use.
• Always use aseptic technique
Laboratory Safety Continued
• Place contaminated materials in
appropriate containers for disposal.
• Never use a mouth pipette.
• WASH HANDS
• REPORT ALL LABORATORY
INCIDENTS IMMEDIATELY!!!!
Equipment and Supplies
• Incubator
• Sterilizing heat source
• Bunsen burner
• Heating element
• Alcohol lamps
• Media
• Microscope
• Antimicrobrial sensitivity discs
• Miscellaneous other equipment:
• Metal loop
• Slides
• Sterile cotton tip applicators
• Wax markers
Incubators
• Allows organism to be grown under
controlled conditions
• Keeps specimen at 37 C (98.6 F)
• Human body temperature and room
air Oxygen
• Most cultures are grown overnight and
held at least 48 hours.
Sterilizing Heat Sources
• Bunsen Burner
• Sterilizes metal loop used for
transferring organisms to be inoculated
into growing media
• Electric heating element
• Usually ceramic, an enclosed heater.
• Eliminates the need for a natural gas
• Alcohol lamps
• Less expensive
• Glass lamp with wick
• Does not sterilize metal loops that
quickly
Media
• Nutritive media- grows all types of bacteria
• Selective media- grows only certain types
of bacteria
• Gram negative or gram positives
• Enriched media- basic nutrient media with
extra nutrients added-blood, serum, or
egg.
• Differential Media- contains elements that
differentiate certain types of bacteria (ex.
Lactose fermenters or hydrogen sulfide
producers)
Media Continued
• Examine media for accidental
bacteria/fungal contaminants before use
• Incubate al plates UPSIDE DOWN
• Prevents condensation from dripping
onto cultures.
• Media may be solid (agar) or liquid (broth).
• Plate is a flat, round container of agar
• Tube is a screw-top container that may
contain broth and agar
• Slant, a tube of agar that has been
allowed to gel at an angle.
Basic Nutrient Media
• Peptone- hydrolyzed protein that can be metabolized by
bacteria (provides AA)
• Principal nutrient of the medium
• Salt
• Dextrose- gives carbon and energy for bacteria
• Water
• Meat extract- provides water soluble CHO nitrogen and
vitamins
• Solidifying agents- Agar, gelatin
• Basic Nutrient Types:
• Nutrient Agar
• Trypticase soy agar (TSA)
Selective Media
• Blood Agar
• Chocolate Agar
Media supports most bacterial pathogens
Blood Agar
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TSA + 5% sheep blood
Can refer to Blood Agar Plate (BAP)
Blood agar should be bright red
Brownish-red color may indicate:
• Blood is too old, RBC’s are hemolyzing
• Blood was added to the agar base
when the medium was too hot
• Inadequate mixing of blood and agar
Blood Agar Continued
• Also acts as a differential media
• Four types of hemolysis:
• Alpha hemolysis- partial hemolysis
• Narrow band of greenish slimy discoloration around the
colonies
• Beta hemolysis- complete hemolysis
• Clear zone around the bacterial colony
• Gamma hemolysis- no hemolysis
• Delta hemolysis- Double zone hemolysis
• Double ring of hemolysis around colonies
• Can differentiate different species of Streptococcus.
Chocolate Agar
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For Hemophilus spp.
A very nutritive media
Hemolyzed RBC’s with growth factors
Has increased Carbon dioxide
MacConkey Media
• Both a selective and differential media
• Selects for Gram-negative
• Uses crystal violet as a gram + inhibitor
• Suppresses growth of gram +
• Indicators
• Lactose and neutral red
• Lactose fermentors (E. coli, Enterobacter, and Klebsiella)
produce acid from lactose and grow as pinkish-red colonies
• Lactose non-fermentors- produce colorless colonies
• Ex. No growth on MAC and good growth on BAP suggests Gram
+
• Inhibits swarming of Proteus
Enriched Media
• Brain-heart Infusion Broth (BHIA)
• Mueller-Hinton agar (MH)
• For antibiotic sensitivity
Brain-heart Infusion Broth (BHIA)
• General purpose broth used to increase
the numbers of organisms before they are
plated
• Ex. Listeria from brain tissue may be
difficult to grow, finely cut brain tissue is
incubated for weeks in BHIA, then
cultured.
Differential Media (several types)
• Urea agar slant
• Will turn from peach to pink with
ammonium production
• Triple sugar iron agar
• Have lactose, sucrose, glucose
• Differentiates- Salmonellas and other
enteric bacteria
• Mannitol salt agar
• For select Staph species
Inoculating Culture Media
• Use sterile technique
• Flame neck of tube when transferring
organisms.
• Do not put the cap down but hold between
last 2 fingers
• Flame the near portion of the wire 1st,
then work towards the contaminated end
• Turns red
• Prevents bacteria splatter
Streaking Plates
• Flame the bacterial loop between and cool
• Each streak is overlapped only 1-2 times
to avoid depositing excessive numbers of
bacteria
• Will give discreet isolated colonies
• Use the entire plate to streak
• Turn plate for each streak
• Flame/cool loop in between
Materials you will need:
• Gloves
• Inoculating loop OR sterilized wooden sticks for streaking
• Permanent marker or grease pencil to label your plates
beforehand
• Bunsen burner or sterilizing heater if intending to sterilize the
inoculating loop between streaks
• Swab for collecting the primary inoculum, if intending to collect
bacteria from an environmental source
• Agar plate
• Incubator, if incubating at a controlled temperature, such as 37｡
C. However, many common microbial species will grow on
plates left at room temperature, through their growth may be
slower than if the plates were incubated at 37｡C
• Antibacterial soap to wash your hands
• 5% bleach solution to clean your work area when finished.
Step One:
(The Primary Streak)
If you are right-handed, hold the plate in your left hand, and the inoculating
loop in your right - as through you would a paint brush. If you are lefthanded, use the opposite hands.
Touch your inoculating loop
(sterile swab, or sterile stick as
shown in the picture) to the
material you want to spread.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Go back and forth a number
of times in a small area of the
Agar plate.
The goal is to spread your
material completely over this
inital area of the plate.
Step Two:
(The Secondary Streak)
Sterilize your inoculating loop, or use a fresh, sterile inoculating stick or swab. Make sure
the loop is cool before your next streak. If you were to use the original loop, you will not
be diluting the individual microbes you applied in the first streak.
Pick up the plate and rotate it 1/4
of a turn to your left (if righthanded), or to your right (if left
handed).
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Run the loop through the
previous streak 2-3 times, then
draw it along 1/3 of the remaining
plate, as shown by the blue line in
the image.
Step Three:
(The Tertiary Streak)
Rotate the plate another 1/4 turn and sterilize yourinoculating loop or
take a fresh, sterile stick or swab. Again, make sure to cool your loop
between streaks.
Run the loop through
the previous, secondary
streak 2-3 times, and
draw the streak over a
remaining 1/3 of the
plate, as shown.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Step Four:
(The Quarternary Streak)
Rotate the plate another 1/4 turn and sterilize the inoculating loop.
Again, cool the loop between streaks, or use a new sterile swab.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Run the loop through
the previous tertiary
streak 2 times and
draw over the
remaining free space
in the plate, being
careful not to contact
the primary streak
(yellow).
Inoculating Slants
• Only the surface
• Streak “S” shaped
• Both surface and butt
• Stab the butt
• Withdraw the wire up same insertion
path
• Then streak the slant
Colony characteristics
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Size- pin point, medium, large
Color- yellow, white, gray, cream, etc.
Density- opaque, transparent
Elevation-raised, flat, convex, droplike
Form-circular, irregular
Consistency- buttery, brittle, sticky
Odor- pungent, sweet, etc
Hemolysis- alpha, beta, gamma
• What is the basic shape of the
colony? For example, circular,
filamentous, etc.
• What is the cross sectional
shape of the colony? Turn the
Petri dish on end.
• What is the magnified shape
of the edge of the colony?
• How does the surface of the colony
appear? For example, smooth,
glistening, rough, dull (opposite of
glistening), rugose (wrinkled), etc.
• For example, transparent (clear),
opaque, translucent (almost clear,
but distorted vision, like looking
through frosted glass), iridescent
(changing colors in reflected light),
etc.
• For example, white, buff, red,
purple, etc.
Bacterial Staining
• Gram’s stain
• Acid fast stain
• Geimsa stain
Examine number, types of bacteria
Gram Staining
• Used to categorize bacteria as Gram + or Gram -.
• Use cell wall morphology
• Use young colonies (24 hours old)
• Older colonies may not give proper results
• Decolorization
• Bacteria that retain the crystal violet-iodine
complex stain Purple are Gram +
• Those that lose the crystal violet and stain Red by
safarin or basic fuschsin are Gram -
Gram Staining
• Obtain a sample from one colony with
sterile wire loop
• Mix with drop of saline or water on the
slide, if from broth use 2-3 loopfuls
• Circle the area with wax pencil
• After drying, heat fix- DO NOT overheat
• Prevents bacteria from washing off
• Kills bacteria and makes them pick up
stain
Gram Staining
• Exam also morphology
• Bacilli
• Cocci
• Coccobacilli
• Spiral
• Paris, chains, clusters
• Sometimes get a Gram variable reaction
• Both Gram + and Gram – on same
organism
Gram variable
• May occur because:
• Excessive heat fixation
• Decolarization
• Overly thick smear
• Old cultures
• Poor quality stain
Potassium Hydroxide Test (KOH)
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Checks the gram reaction
Place a loopful of 3% KOH on slide
Place a large amount of surface growth
Stir, then slowly lift loop up after 30
seconds
• Gram neg- sticky strand
• Grand +- does not form a strand on lifting
Size/shape/arrangement
• Cocci- spherical in shape
• Staph aureus
• Bacillus- rod in shape
• Bacillus anthracis
• Spiral• Loose spirals- Borrelia
• Tight spirals- Leptospira
• Comma shaped spiralsCampylobacter
Bacterial arrangement
• Single- most bacilli
• Pairs- Streptococcus
pneumoniae
• Clusters- Staph aureus
• Chains- Strep species
• Palisades- Chinese letter
pattern- Corynebacteria
Acid Fast stain
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Several types of stains
Used to detect Mycobacteria and Nocardia
Acid fast bacteria- stain red
Non acid fast- stain blue or green
• Depends on stain used- brilliant green
or methylene blue
Giemsa stain
• Used to detect spirochetes and ricketssiae
• Demonstrates the capsule of Bacillus
anthracis
Procedure for Identifying Bacteria
• Streak on Blood agar and MAC
• Incubated 18-24 hours
• Select colonies from BAP vs. MAC
• MAC may inhibit some colonies
• Gram stain
• Differential media
Gram Positive Cocci
• Staphylococci
• Steptococci
• Micrococci
Catalase Test
• Do when have a gram positive cocci and small gram positive
bacilli (any gram positive colony)
• Tests for the enzyme catalase, which acts on hydrogen
peroxide to produce water and oxygen.
• Place a small amount of an isolated colony from a blood
agar plate on slide and a drop of catalase reagent (3%
hydrogen peroxide).
• Catalase positive- gas bubbles are produced
• Catalase negative- no gas bubbles are produced
• Do not transfer any blood agar with colony because may
get a slightly positive reaction
Coagulase Test
• Do when have a catalase positive, Gram
positive cocci
• Some Staph species have an enzyme that
coagulate plasma.
• In general, the coagulase positive staph
are more pathogenic
• Staph aureus and Staph intermedius are
coagulase positive
• Staph epidermidis is coagulase negative
Tube Coagulase test
• Tube coagulase test- lypophilized plasma
is placed in a test tube with a loopful of
colony
• Positive reaction- has clots after 4
hours
• Slide coagulase test- loopful of colony is
emulsified in a drop of saline. A drop of
rabbit or human plasma is added
• Positive reaction- clumping in 5-20
seconds
Staphylococci
• Gram positive cocci
• Staph aureus- often in grape-like clusters
• Catalase test positive
• +/- Coagulase positive
• Staph aureus is coagulase positive
• Staph aureus- abcesses, wound infections, mastitis
• Staph epidermidis- usually non-pathogenic skin
• Staph intermedius- Skin infections
• Staph hyicus- greasy pig disease, exudative skin lesions
Streptococci
• Gram positive cocci
• Pairs- Strep penumoniae
• Chains
• Catalase negative
• Note hemolysis in blood agar
• Several strep species may be responsible for many illnesses
including pneumonia, mastitis, septicemia, and enteritis.
• Several strep species may cause neonatal septicemia, urinary
infections, pneumonia
• Strep fecalis- (enterococcus). Opportunistic pathogen found in the
GI tract
• Strep equi- Strangles (pus, abscesses, enlarged lymph nodes)
• Other strep equi subgroups may cause mastitis, abortion and
abscesses.
Gram negative Cocci
• Moraxella bovis- large gram negative cocci
that resembles fat rods (coccobacillus)
• Causes pink eye in cattle
(Keratoconjunctivitis)
• Neisseria spp. Often found in respiratory
tract of many normal animals
• N. gonnohoeae- human gonnorhea
Gram positive rods
• Aerobic and anaerobic Gram + rods
• Anaerobic- need specific collection
device
• THIO (thiglycollate broth) grows
anaerobes
• Some Gram + rods produce spores
• Bacillus and Clostridium are
sporeformers
• Spores vary in shape, size, and location
• Non-staining bodies on the Gram stain
Spores (Endospores)
• Central: Bacillus anthracis
• Subterminal: Present near the end
• Terminal: Present at the end
• Clostridium tetani
Gram positive Rods species of
concern
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Bacillus anthracis- causes sudden death in cattle and
sheep (Fatal septicemia), in humans will cause skin and
lung lesions
Bacillus cereus- can cause food poisoning
Bacillus piliformis- acute fatal enteritis in rodents and
foals
Clostridium botulinum- botulism
Clostridium Chauvoei- Blackleg (gas gangrene)
Clostridium perfringens- gangrenous necrosis of the skin,
entertoxemia/pulpy kidney disease of sheep
Clostridium tetani- tetanus
Corynebacterium equi- foal pneumonia
Corynebacterium pseudotuberculosis- casseous
lymphadneitis in sheep
Corynebacterium renale- UTI in cattle
Gram negative Rods
• Grow on MAC
• Note the lactose reaction of the colony
• Do oxidase test if non-lactose fermentor
• All lactose fermentors are oxidase
negative
• Enteric (gut bacteria) pathogens and non
pathogens (opportunistic)
Oxidase test
• Oxidase reagent is added to filter paper
with colony sample
• Positive will change to purple color in
60 sec.
• Oxidase test can differentiate between
gram negative bacteria within 2 groups
• Pseudomonas- oxidase positive
• E. coli- oxidase negative
Gram Negative Rods
Oxidase Positive
Oxidase Negative
Patuerella
Enterobacteriaceae
Bordetella
E. Coli
Pseudomonas
Shigella
Actinobacillus
Salmonella
Hemophilius
Proteus
Gram Negative Coccobacilli
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May be difficult to tell from cocci
May not grow on MAC
Moraxella
Brucella• Brucellosis canis- abortion,
diskosponylitis
• Brucella abortus- cattle
Higher/Resistant Bacteria
• Mycobacterium
• Mycobacterium tuberculosispneumonia in humans and primates
• Mycobacterium avium- GI/respiratory
infections in birds
• Mycobacterium paratuberculosisJohne’s disease of cows, sheep and
goats
• Nocardia
• Purulent lesions in dogs/cats
• Long branching filamentous rods
Spirochetes
• Leptospira
• Campylobacter
Antimicrobial Susceptibility
Testing
• Performed when bacteria are isolated from
a patient
• Determines the susceptibility or resistance
to antibiotics
• Ideally, the specimen used for the
susceptibility testing should be collected
prior to treatment with antibiotics.
• Must isolate the suspected pathogen
• Gram stain• Some antibiotics are more effective
against Gram + or Gram - bacteria
Terminology
• Antibiotic- a biologic substance developed from a
microbe that is either bacteriostatic or bacteriocidal.
Most antibiotics are produced from molds, but a
few are produced from bacteria.
• Bacteriocidal- kills bacteria
• Bacteriostatic- drug inhibits a bacteria’s growth but
does not kill the bacteria
• Antibiotic resistance- the aquired ability for a
bacteria to grow in the presence of an antibiotic.
• Antibiotic therapy aims to treat infection with a drug
that the causative bacterial pathogen is sensitive
to.
Antimicrobrial Susceptibility
Testing
• Agar diffusion method
• Most commonly used
• Uses paper discs impregnated with antimicrobials
• Requires measurement of zone sizes to give an
estimate of the antibiotic susceptibility (zone of
inhibition)
Kirby-Baur Antimicrobial Testing
Method
• Uses Mueller-Hinton blood with or without
5% blood
• Make sure there is no gross moisture on
the agar surface or lid before use.
• Grows most bacteria, but with Strep,
usually have to use the blood additive
Kirby- Bauer Method: Preparation
of Bacteria
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Preparation of the bacteria
• 1. Need to standardize the bacterial sample
• 3-4 well isolated colonies are obtained with sterile loop and
placed in 3 ml tube of sterile saline
• 2. Inoculate the Mueller-Hinton agar with the suspension using
sterile cottonswab
• Be sure to streak the entire plate evenly in several directions
• Streak the plate 3 times at 120 degree angles
• Allow to dry for 15-30 minutes
• 3. Application of antibiotic paper discs
• Using a disk dispenser, apply the disk to the agar surface
• Apply slight pressure with sterile forceps to ensure the disks
will adhere to the surface
• 4. Incubate for 18-48 hours
• 5. Measure the zones of inhibition with mm ruler
Kirby-Bauer Method: Reading
Zones of Inhibition
• Read the zone size from the top surface
with the lid of the plate removed
• Zone sizes are divided into 2 major
categories
• Resistant
• Susceptible
• Intermediate susceptibility implies
that at higher dosages the bacteria
may be susceptible
Abscesses
• Small Animals
• Staph aureus
• Steptococci
• Pastuerella multocida (from bite
wounds)
• Pseudomonas aeruginosa
• Large Animals
• Corynebacterium pyogenes
• Corynebacterium pseudotuberculosis
• Strep
• Staph and Pseudomonas
Lumpy Jaw and Wooden Tongue
• Infections caused by Actinomyces bovis
and Actinobacillus lignieresi in cattle
• Abscesses in the jaw region
• Has granules in the pus
Other diseases of interest
• Brucellosis
• Causes abortions in many species
• Brucella abortus, canis, ovis, suis
• Mastitis
• Staph aureus, strep agalactiae, e.coli,
corynebacterium, pseudomonas, pastuerella
• UTI (small animal)
• E. coli, proteus, staph aureus, pseudomonas,
enterobacter
• Bordetella
• Bordetella bronchiseptica