1. Introduction to Microbiology
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2. Microbiology Basics
A basic understanding of the characteristics of bacteria, fungi, and viruses will aid the veterinary technician in collection, handling, and evaluation of samples
Identification of bacterial, fungal, and viral pathogens is the primary purpose of microbiologic examinations
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3. Bacterial Cell Morphology
Prokaryotic – no nuclear membrane
Most cellular organelles are absent, except cell walls, plasma membranes, and ribosomes
Some may contain capsules and flagella and can develop endospores
Small – 0.2 to 2.0 mcg
Have requirements for temperature, pH, oxygen tension, and nutrition
Considered when collecting and preparing samples
Used to help identify samples
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4. Oxygen Requirements Obligate aerobes – require O2 to survive
Obligate anaerobes – growth inhibited or killed in the presence of O2
Facultative anaerobes – can survive in O2 but growth is limited
Microaerophilic – prefer reduced O2 tension
Capnophilic – require high levels of carbon dioxide
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5. Other Requirements pH – most live in the range of 6.5 to 7.5
Nutritional
Fastidious microbes – strict nutritional requirements
Agar medium
Temperature
Mesophiles – nearly all pathogenic bacteria in animals grow best at 20° to 40° C
Psychrophiles – lower temperatures
Thermophiles – higher temperatures
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6. Morphology Classified by shape and arrangement
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7. Morphology (cont.) Classified by arrangement
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8. Spores Some genera of bacteria have endospores
Resistant to heat, desiccation, chemicals, and radiation
Location in cell helps classify
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9. Bacterial Growth
Contain a single DNA strand and reproduce primarily by binary fusion
4 phases
Initial – lag phase – adapting to new media
Exponential – doubling or rapid growth
Continues until nutrients are used up, waste products accumulate, or space is limited
Stationary – no net increase or decrease
Final – death phase
Spore formation occurs during this phase
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10. Bacterial Growth (cont.)
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11. Fungal Characteristics
Heterotrophs
Parasitic or saprophytic (decomposers)
Most multicellular – except yeast
Eukaryotic cells with cell walls of chitin
Large webs (mycelium) of slender tubes (hyphae)
Grow toward food sources
Digest food internally – enzymes
Yeasts reproduce by budding
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12. Laboratory Safety Potentially pathogenic and/or zoonotic
Aseptic technique
Personal protective equipment
Long sleeves, knee-length white laboratory coat or clean surgical scrubs
Prevent contamination of street clothes
Gloves
Face masks
Eye protection
Wash hands
Autoclave or dispose of properly
Treat spilled cultures with disinfectant – 20 minutes
No eating, drinking, handling contact lenses, or cosmetics
Hair pulled back
Report accidents promptly
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13. Equipment and Supplies
A good-quality incubator
Collection and preparing supplies
Sterile cotton-tipped swabs
Dull scalpel blades
3- to 20-mL syringes and 21- to 25-G needles
Sterile endotracheal tube or jugular or urinary catheter
Collection tubes and preservatives
Rayon swabs in transport media
High-quality glass slides and coverslips
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14. Equipment and Supplies (cont.)
Inoculating loops or wires – reusable metal or disposable plastic
Bunsen burner or alcohol lamp
Candle jar or anaerobe jar
Variety of culture media
Antibiotic disks and dispensers
Gram stains and other stains
Scissors, forceps
Discard jar – disinfectant
Wooden tongue depressors
Racks for tubes
Refrigerator – cold packs and polystyrene shipping containers
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15. Equipment and Supplies (cont.)
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16. Culture Media
Any material, solid or liquid, that can support the growth of microorganisms
Agar is dried extract of sea algae
Gelatin is a protein obtained from animal tissues
Store refrigerated at 5° to 10°C
Away from inside wall to avoid freezing
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17. Types of Media Six general types
Transport
Keep microbes alive but do not encourage growth
General purpose
Enriched
Meet requirement of fastidious pathogens
Selective
Contain antibacterial substances
Differential
Allow bacteria to differentiate into groups by biochemical reactions
Enrichment
Favor growth of a particular group or organism
Some medias contain more than one type
Hundreds of different types of culture media, but veterinary medicine uses only a few
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18. Commonly Used Media
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19. Blood Agar Enrichment and a differential media
Trypticase soy agar with sheep blood – most common
Four distinct types of hemolysis can be detected
Alpha – partial hemolysis creates a band of greenish or slimy discoloration around the colony
Beta – complete hemolysis creates a clear zone around the colony
Gamma – hemolysis that produces no change in the appearance of the medium
Delta – double zone hemolysis
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20. Alpha Hemolysis
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21. MacConkey Agar and Eosin-Methylene Blue Agar
Selective and differential media
MacConkey contains:
Crystal violet (suppress growth of Gr + bacteria)
Bile salts – selective for lactose-fermenting Enterobacteriaceae and a few gram bacteria
EMB – performs the same function as MacConkey agar
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22. MacConkey Agar Indicators are lactose and neutral red
Lactose-producing bacteria grow as pinkish-red colonies
Nonlactose-fermenting bacteria produce colorless colonies
Inoculation of both blood and MacConkey agar can yield information
No growth on MacConkey but good growth on blood agar suggests gram-positive bacteria
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23. Thioglycollate Broth
Liquid medium used to culture anaerobic bacteria to determine O2 tolerance of microbes
Contains a stable O2 gradient with high O2 near surface and anaerobic conditions near the bottom
Obligate aerobes will only grow in the top layer
Obligate anaerobes grow in the lower portion
Facultative – throughout the medium but best in middle of tube
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24. Urea Tubes
Urea slants are streaked with inoculum and incubated overnight at 37° C
Urea medium is peach colored
If bacteria hydrolyze, the urea in the medium, ammonia production turns the medium a pink color
Negative, no color change
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25. Mannitol Salt Agar Not routinely used
Highly selective medium for Staphylococci
Isolate Staphylococcus aureus
Contains
High salt level, mannitol, and pH indicator phenol red
Staphylococci – salt tolerant
S. aureus – ferments mannitol
S. aureus colonies surrounded by medium yellow
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26. Mueller-Hinton General purpose
Performance of the agar diffusion antimicrobial sensitivity test
Chemical composition does not interfere with diffusion of antimicrobials through agar
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27. Dermatophyte Test Media
Most common standard dermatophyte test medium (DTM)
Contains an indicator that turns red if dermatophytes are present
Rapid sporulation medium (RSM) or enhanced sporulation medium (ESM)
Color indicators
Contain Sabouraud agar and antimicrobials
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28. Quality Control Cultures
Procedures and supplies monitored for quality and accuracy
Antibacterial susceptibility tests
Media
Biochemical tests
Identification tests
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29. Culturing Specimens
A systematic approach is needed to identify pathogenic bacteria
Develop flowcharts for use in the clinic for the most commonly seen bacteria
Include tests to differentiate those bacteria
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30. Presumptive Identification
Staining and culturing
Veterinarian may start treatment based on results
Plate suspected pathogens on primary medium
Blood agar or MacConkey agar
Incubate for 18 to 24 hours – examine for growth
Follow flow chart for further identification
Definitive identification
Requires additional biochemical testing
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31. Presumptive Identification (cont.)
Blood agar
Grows both gram-positive and gram-negative
Selection of colonies is preferable
MacConkey agar
Gram-positive organisms do not grow
May inhibit growth of potentially pathogenic bacteria
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32. Inoculation of Culture Media
Aseptic technique at all times
Sear surface of cadaver or excised organs
Culture plates kept closed except when removing specimens
Tubes – pass neck of tube through a flame and do not put down cap, hold in pinkie finger
Flame wire – start at near end first and work toward distal end
Could cause splattering of bacteria – aerosolization
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33. Inoculating with a Swab
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34. Quadrant Streaking Method
Designed to aid in isolation of pure bacterial cultures
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35. Inoculations of Tube Media
Enough bacteria should be on the wire to inoculate the surface even after stabbing the butt
Slant surface inoculated in an “S” shape
Tube cap on loosely
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36. Inoculations of Tube Media (cont.)
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37. Inoculations of Cultures
Pathogens that can invade internal organs
Incubate at 37º C
Some fish, skin pathogens, and environmental organisms
Incubate lower than 37º C
Incubation time depends on the generation time of the species and type of medium
Most are 48 hours with examination at 18 to 24 hours
Incubate plates inverted to prevent condensation on the surface of the plate
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38. Colony Characteristics
Help identify the bacterium
Size
Pigment
Density (opaque, transparent)
Elevation (raised, flat, convex, droplike)
Form (circular, irregular, rhizoid, filamentous, undulate)
Texture (glassy, smooth, mucoid, buttery, brittle, sticky)
Odor (pungent, sweet, etc.)
Any hemolysis (alpha, beta, gamma)
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39. Colony Characteristics (cont.)
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40. Colony Characteristics (cont.)
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41. Culture of Anaerobes Do not use swabs
Preferred blocks of tissue (2-inch cube minimum) in a closed, sterile container and pus and exudate collected in a sterile syringe
Air expelled, needle plugged
Special anaerobic specimen collection systems
Culture as soon as possible after collection
Blood agar and thioglycollate broth
Incubate in an anaerobe jar
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42. Stains A variety of stains available Most common
Gram stain
Ziehl-Neelsen (acid-fast) stain
Stain before culturing
Rapid identification
Determine appropriate medium
Determine appropriate antibacterials
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43. Staining Kits
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44. Stains (cont.) Simple stains
Crystal violet and methylene blue
Typically used for yeasts
Lactophenol cotton blue
Confirms identity of fungal organisms
Many stains available but most are performed only in large reference or research laboratories
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45. Gram Stain Used to categorize bacteria Requires four steps
Gram-positive or gram-negative
Based on cell wall structure
Requires four steps
Primary stain, a mordant, a decolorizer, and a counterstain
Mordant – fixes dyes to the structures (cell wall)
Primary stain usually crystal violet
Mordant – Gram’s iodine solution
Decolorizer – 95% ethanol or acetone
Counterstain – basic fuchsin or safranin
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46. Gram Stain Procedure Thin sample applied to slide
Swabs can be gently rolled on slide
Sterile wire to one young colony (24 hour)
Older colonies may not stain accurately
Mix sample from plates with a drop of water or saline
If broth – 2 to 3 loopfuls
If liquid – circle drop area with a wax pencil
No matter source – take care not to damage organisms
Air dry then heat fix by passing through a flame 2 to 3 times, specimen side up
Do not overheat – warm not hot
Prevents sample from washing off, preserves morphology, kills the bacteria, and renders them permeable to the stain
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47. Gram Stain Interpretation
Gram-positive
Bacteria that retain the violet-iodine complex stain purple
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48. Gram Stain Interpretation (cont.)
Gram-negative
Lose the crystal-violet or purple color and stain red
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49. Ziehl-Neelsen Stain Primarily used to detect acid-fast organisms
Mycobacterium and Nocardia species
Many stains available, but few work well in veterinary practice laboratories
Several steps
Primary stain – dimethyl sulfoxide (DMSO) and carbol fuchsin
Decolorizer – acid-alcohol
Counterstain – methylene blue
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50. Ziehl-Neelsen Stain Procedure
Slide is air dried and heat fixed
Primary stain is flooded on slide
Slide heated over flame until stain steams
Cool slide for 5 minutes, then rinse with tap water
Acid alcohol is used to decolorize – 1 to 2 minutes until red color is gone
Rinse slide
Counterstain added then rinsed with water and air dried
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51. Ziehl-Neelsen Stain (cont.)
Agents like DMSO allow the stain to penetrate stain-resistant cells such as Mycobacterium
If stain isn’t removed by acid alcohol then the organism is “acid-fast” and appears red
Non–acid-fast cells stain blue
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52. Ziehl-Neelsen Stain (cont.)
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53. Giemsa Stain
Used to detect spirochetes and rickettsiae and the capsule of Bacillus anthracis and the morphology of Dermatophilus congolensis
Smear fixed with absolute methanol for 3 to 5 minutes and air dried
Dip in diluted stain for 20 to 30 minutes
For Borrelia anserina – the smear is gently heated while covered with the stain for 4 to 5 minutes then rinsed and air dried
Purplish-blue-stained bacteria
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54. Specialized Stains
Flagella, capsule, and endospore stains are available but have limited application in the average veterinary practice
Expensive stains
Malachite green endospore
stain of Bacillus
anthracis
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55. Antimicrobial Sensitivity
Performed to determine the susceptibility or resistance to specific antimicrobial drugs
Helps veterinarian choose the most appropriate antimicrobial for the patient
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56. Agar Diffusion Method Most commonly preferred method
Uses paper disks impregnated with antimicrobials
Quantitative and requires the measurement of inhibitory zone sizes
Gives an estimate of susceptibility
Concentrations on disks correlate with therapeutic levels of the drugs in tested animals
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57. Agar Diffusion Method (cont.)
Modified Kirby-Bauer technique
U.S. Food and Drug Administration method
International collaborative study
Minimum inhibitory concentration (MIC)
The smallest concentration that can inhibit growth
Disks with varying concentrations
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58. Antimicrobial Disks Kept in the refrigerator when not used
Outdated disks should not be used
Potency of disks monitored with control organisms
A disk dispenser for placement obtained from the manufacturer of the disks
Caliper for measuring
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59. Antimicrobial Disks (cont.)
Inoculation of a thioglycollate or trypticase soy broth tube at the time the plates are streaked is recommended
In case the in-house culture is inconclusive
Broth cultures can be sent to a reference lab for confirmation
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60. Indirect Sensitivity Testing
Requires that colony samples be taken from a culture plate and subcultured in broth media
Incubate to achieve turbidity to match a standardized 0.5 McFarland suspension
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61. Direct Testing Method
Application of undiluted samples (urine) directly to a Mueller-Hinton plate
Not as precise
Reasonable results if only one organism is present
Interpret with caution if multiple organisms are present
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62. Disk Placement Dispenser or sterile forceps 10 to 15 mm apart
Flamed and cooled between each use
10 to 15 mm apart
Avoid overlapping of ZOI
Tamp disk into the agar with a sterile swab
Incubate, inverted and aerobic at 37º C, within 15 minutes of disk placement
Stacks of four or fewer
If taller, the middle plates take longer to get to temperature
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63. Zone of Inhibition
Measurement is compared to a chart of ZOI to determine the relative resistance of the bacterium to the antimicrobial
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64. Reading of ZOI Plates read after a constant time period
Usually 18 to 24 hours
Prolonged incubation may alter the size of ZOI
Zones read from the bottom of the plate using a caliper, transparent ruler, or template
Measured to the nearest millimeter
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65. Interpretation of Zone Sizes
Zone sizes are divided into two major categories
Resistant
Susceptible
Implies the organism is susceptible to ordinary doses of antimicrobials
Intermediate susceptibility
Implies the organism is susceptible to ordinary doses of antimicrobials when the drug is concentrated in the urine or tissues
Or the drug may be used for treatment of systemic infections if a high dose is safe
Size not indicative of efficacy
Some drugs do not diffuse through agar
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66. Control Organisms Susceptible reference organisms
S. aureus (ATCC 25923)
E. coli (ATCC 25922)
Should be tested regularly in parallel with each batch of antimicrobial susceptibility tests
Used to check growth-supporting capability of medium, potency of disks, and other factors
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67. Colony Count
The presence of pathogenic bacteria does not necessarily indicate infection
Colony counts on cultured urine can help support a diagnosis of UTI
After incubation all colonies are counted and multiplied by 100 to determine the number of colony-forming units per millimeter of urine
Guideline for significance
>1000 CFUs in cystocentesis sample
>10,000 CFUs in catheter sample
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