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

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, RBC’s 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.

16. Chocolate Agar For Hemophilus spp. A very nutritive media
Hemolyzed RBC’s 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 Gram’s 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 -

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 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

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- Johne’s 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 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.

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 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 hours
5. Measure the zones of inhibition with mm ruler

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