Microbiology

1
Microbiology

• Clinical Pathology

2
Microbiology

• The study of microscopic organisms
• Clinical microbiology is the identification of
  these organisms that cause clinical illness

3
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

4
Laboratory Safety Continued

• Place contaminated materials in appropriate
  containers for disposal.
• Never use a mouth pipette.
• WASH HANDS
• REPORT ALL LABORATORY INCIDENTS IMMEDIATELY!!!!

5
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
• Stain

6
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.

7
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

8
(No Transcript)
9
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)

10
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.

11
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)

12
Selective Media

• Blood Agar
• Chocolate Agar
• Media supports most bacterial pathogens

13
Blood Agar

• 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, RBCs are hemolyzing
• Blood was added to the agar base when the medium
  was too hot
• Inadequate mixing of blood and agar

14
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.

15
(No Transcript)
16
Chocolate Agar

• For Hemophilus spp.
• A very nutritive media
• Hemolyzed RBCs with growth factors
• Has increased Carbon dioxide

17
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

18
Enriched Media

• Brain-heart Infusion Broth (BHIA)
• Mueller-Hinton agar (MH)
• For antibiotic sensitivity

19
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.

20
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

21
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

22
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

23
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.

24

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
left-handed, 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. 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.
25
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 right-handed), or to your right (if
  left handed).
• 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.

26
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.

27
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.

• 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).

28
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

29
Colony characteristics

• 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

30
Form

• What is the basic shape of the colony? For
  example, circular, filamentous, etc.

31
Elevation

• What is the cross sectional shape of the colony?
  Turn the Petri dish on end.

32
Margin

• What is the magnified shape of the edge of the
  colony?

33
Surface

• How does the surface of the colony appear? For
  example, smooth, glistening, rough, dull
  (opposite of glistening), rugose (wrinkled), etc.
  

34
Opacity

• For example, transparent (clear), opaque,
  translucent (almost clear, but distorted vision,
  like looking through frosted glass), iridescent
  (changing colors in reflected light), etc.

35
Chromogenesis (Pigmentation)

• For example, white, buff, red, purple, etc.

36
Bacterial Staining

• Grams stain
• Acid fast stain
• Geimsa stain
• Examine number, types of bacteria

37
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 -

38
(No Transcript)
39
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

40
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

41
Gram variable

• May occur because
• Excessive heat fixation
• Decolarization
• Overly thick smear
• Old cultures
• Poor quality stain

42
Potassium Hydroxide Test (KOH)

• 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

43
Size/shape/arrangement

• Cocci- spherical in shape
• Staph aureus
• Bacillus- rod in shape
• Bacillus anthracis
• Spiral-
• Loose spirals- Borrelia
• Tight spirals- Leptospira
• Comma shaped spirals- Campylobacter

44
Bacterial arrangement

• Single- most bacilli
• Pairs- Streptococcus pneumoniae
• Clusters- Staph aureus
• Chains- Strep species
• Palisades- Chinese letter pattern- Corynebacteria

45
Acid Fast stain

• 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

46
Giemsa stain

• Used to detect spirochetes and ricketssiae
• Demonstrates the capsule of Bacillus anthracis

47
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

48
Gram Positive Cocci

• Staphylococci
• Steptococci
• Micrococci

49
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

50
(No Transcript)
51
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

52
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

53
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

54
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.

55
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

56
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

57
Spores (Endospores)

• Central Bacillus anthracis
• Subterminal Present near the end
• Terminal Present at the end
• Clostridium tetani

58
Gram positive Rods species of concern

• 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

59
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)

60
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

61
Gram Negative Rods
Oxidase Positive Oxidase Negative
Patuerella Enterobacteriaceae
Bordetella E. Coli
Pseudomonas Shigella
Actinobacillus Salmonella
Hemophilius Proteus
62
Gram Negative Coccobacilli

• May be difficult to tell from cocci
• May not grow on MAC
• Moraxella
• Brucella-
• Brucellosis canis- abortion, diskosponylitis
• Brucella abortus- cattle

63
Higher/Resistant Bacteria

• Mycobacterium
• Mycobacterium tuberculosis- pneumonia in humans
  and primates
• Mycobacterium avium- GI/respiratory infections in
  birds
• Mycobacterium paratuberculosis- Johnes disease
  of cows, sheep and goats
• Nocardia
• Purulent lesions in dogs/cats
• Long branching filamentous rods

64
Spirochetes

• Leptospira
• Campylobacter

65
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

66
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 bacterias 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.

67
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)

68
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

69
Kirby- Bauer Method Preparation of Bacteria

• 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

70
(No Transcript)
71
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

72
Abscesses

• Small Animals
• Staph aureus
• Steptococci
• Pastuerella multocida (from bite wounds)
• Pseudomonas aeruginosa
• Large Animals
• Corynebacterium pyogenes
• Corynebacterium pseudotuberculosis
• Strep
• Staph and Pseudomonas

73
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

74
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