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Diagnostic Microbiology. Reference: Laboratory Procedures for Veterinary Technicians 5 th ed (Hendrix

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Diagnostic Microbiology

Reference: Laboratory Procedures for Veterinary Technicians 5thed

(Hendrix & Sirois)

Microbiology: The study of microbes

___________________: organisms too small to be seen with the naked eye

Bacteriology, mycology, and virology are the studies of bacteria, fungi, and viruses, respectively.

Most microbes found on and in the body are ________________________ (i.e. normal flora)

Samples collected from locations, such as the spinal column, blood, and the urinary bladder should be free of normal flora.

Microbes considered normal flora and nonpathogenic when found in one location can produce significant disease in a site where they should not be found.

Culture Media
  • Culture media: any material, solid or liquid, that can support the _________ of a microorganism.
    • Available as dehydrated powder or as prepared agar plates or ready-to-use liquid media for biochemical tests.
    • Solidifying agents used in preparing solid media include ___________ and ________________.
      • Agar - dried extract of sea algae known as agarphytes
      • Gelatin – protein obtained from animal tissues.
      • Store agar plates refrigerated at 5⁰ C to 10⁰ C and away from internal walls of refrigerator.
Mycology

Fungi are___________________(organisms unable to synthesize metabolic products from inorganic materials; they must rely on an organic source of carbon that has originated as a part of another living organism) and may be_______________or __________________________.

Most are multicellular (except for yeasts) and are ________________(having a true nucleus) cells with cell walls composed of_____________.

Fungal organisms consist largely of webs of slender tubes called________________, that grow toward food sources.

Mycology

Fungi digest food externally, through release of digestive enzymes, and then bring the resulting small molecules into the hyphae.

Hyphae make up a branching web called a________________________.

Fungal organisms may also have a reproductive structure called a_______________________ that produces and releases reproductive cells called__________________.

Mycology
  • Different groups of fungi produce different types of spores.
    • Yeasts reproduce by ___________________ rather than by spore formation.
  • Most fungi rely on sexual and asexual reproductive systems
  • Asexual spores produced by some fungi are sporangiosporesor conidia.
  • Sexual spores include ascospores, basidiosporesand zygospores.
Fungal Terminology

Macroconidia

Microconidia

Hyphae

Mycelium is a web of hyphae

Dermatophytes

Dermatophytesare ____________________ (keratin seeking) fungi that invade hair, nails, and superficial layers of skin. Because of the nature of the lesion, it is also referred to as ________________.

They are considered ____________________ due to the nature of the tissue in which they invade.

Dermatophytes are composed of more than three dozen organisms in the taxonomic genera Microsporumand Trichophyton.

The three most commonly seen species are ____________________ canis, M. gypseum, and _____________________________________.

Dermatophyte Testing
  • Some dermatophytes can be visualized microscopically by mounting a few plucked hairs in a few drops of 10% potassium hydroxide (can add dimethyl sulfoxide) then applying a coverslip and examining microscopically after 2 to 10 min. for small globular arthrospores attached to hair shafts.
  • A _________________________ may be used to screen suspect lesions.
    • Some species of Microsporummay fluoresce a clear apple green under the lamp in a darkened room.
Dermatophyte Testing Products
  • Several products available for culturing dermatophytes.
  • Most common is standarddermatophyte test medium (DTM)
    • An indicator that turns _________ in the presence of most dermatophytes
  • Rapid sporulationmedium (RSM) or enhanced sporulation medium (ESM) and Standard Sabouraud dextrose agar are also available.
Dermatophyte Culture MediaDermatophyte Testing Procedure
  • Clean skin lesion to remove surface contamination and collect specimens from lesion periphery.
    • Broken hair shafts and dry scale most likely to contain viable organisms.
  • Push specimens into and partially below the surface of the media and incubate the culture at room temperature with the cap or plate cover loosened; observe daily for growth. (Usually x10-14 days: longer can cause false positive)
  • Examine any growth microscopically with clear cellophane tape and lactophenol cotton blue stain to confirm the presence of pathogenic forms.
MicrosporumcanisMicrosporumgypseumTrichophytonmentagrophytesNon-Dermatophytes and Testing
  • The three most important systemic mycoses are coccidioidiomycosis, histoplasmosis, and blastomycosis.
    • Dimorphic fungi like Blastomycesand Histoplasma spp. grow as yeasts at body temperature and as molds at 25⁰ C.
    • Characteristics of systemic dimorphic fungi of veterinary importance are listed in table 4-7.
    • Tissue sections showing invasion may be needed for definitive diagnosis of mycotic infection.
    • These are serious zoonotic agents; therefore the small lab should not attempt to isolate and culture them.
Yeast
  • There are only a few clinical situations in which yeasts are significant veterinary pathogens.
    • Malassezia _________________________ is often found in cases of ________________________________, and is an emerging cause of seborrheic and hypersensitivity reactions associated with dermatitis. Observed in smears of exudate stained as monopolar budding yeast.
    • Candida albicans is a common opportunistic fungal pathogen involving ________________ membranes. Direct microscopic examination reveals unicellular budding yeast without a capsule.
    • Other yeasts are isolated much less frequently.
Virology
  • Virologic techniques include histopathologic and serologic examination, electron microscopy, and attempted isolation and identification of the virus.
  • Serologic tests are available for most viral diseases.
  • Rising antibody titer indicates recent infection by the virus.
  • Virus isolation is expensive and time consuming and may provide a diagnosis only after the animal has recovered or died.
    • Is most successful when specimens are collected early in the active infectious phase.
Virology
  • Viruses vary greatly in ability to remain viable in tissues and exudates
    • Often present in the nasal or pharyngeal secretions early in the acute stage of respiratory diseases
  • Viral diseases often are complicated by pathogenic bacteria acting as secondary invaders.
  • Samples for virology testing must be collected aseptically, kept at 4⁰ C (39.2° F), and taken to the laboratory as quickly as possible.
Bacterial Morphology
  • Bacteria: prokaryotic cells that range in size from 0.2 to 2.0 micrometers
  • Most cellular organelles are absent except: cell walls, plasma membranes, and ribosomes
  • Bacteria have specific requirements for temperature, pH, oxygen tension, and nutrition
  • Majority of clinically significant bacterial species require a pH of 6.5 to 7.5.
Bacterial Morphology

Obligate ______________: bacteria that require oxygen to survive.

Obligate ___________________: bacteria killed in the presence of oxygen or whose growth is inhibited in the presence of oxygen

Faculative anaerobes: bacteria that can survive in the absence of oxygen but with limited growth.

_______________________: prefer reduced oxygen tension.

____________________: require high levels of CO2.

Bacteria Requirements
  • __________________ requirements vary among bacteria
    • Affect the type of culture media chosen
    • ____________________ microbes have very strict requirements
  • ______________________________ requirements
    • Nearly all pathogenic bacteria grow best at 20 - 40⁰ C
      • referred to as ___________________
    • Bacteria with lower and higher temperature requirements referred to as psychrophiles and thermophiles, respectively.
Bacterial Morphology

Bacteria are organized into four groups according to shape.

Coccus (cocci) – _________________ cells

Bacillus (bacilli) – _________ or cylinders

Spiral – usually occur singly and can be subdivided into loose, tight, and comma shaped

Pleomorphic– shape ranging from cocci to rods

Bacterial Arrangements

Some occur singly, such as spirillaand most bacilli .

Some occur in pairs (diplococci)

Some occur in clusters, bunches, or groups

Some can be arranged in a palisade or a “Chinese Letter” pattern

Bacterial Endospores

A few genera of bacteria form intracellular refractile bodies called endospores or, more commonly, spores.

Organisms in the genera Bacillus and Clostridium are spore formers.

Bacterial spores are resistant to ____________, desiccation, __________________, and radiation.

Bacterial Endospores
  • Spores vary in size, shape, and location in the cell and may be subclassified:
    • ________________: present in the center of the cell, such as Bacillus anthracis.
    • _____________________: present near the end of the cell, such as Clostridium chauvoei.
    • ________________: present at the end or pole of the cell, such as Clostridium tetani.
  • Performing a special spore stain may not be necessary because the endospores can usually be visualized as non-staining, bodies with Gram stain.
Bacterial Endospores

Central

Subterminal

Terminal

Bacterial EndosporesClostridium EndosporesBacterial Growth

Bacterial cells contain a single DNA strand and reproduce primarily by_____________________.

Bacterial growth proceeds through four distinct phases: lag phase, exponential growth phase, stationary phase, and logarithmic decline phase.

Rate of growth during exponential growth phase often referred to as doubling timeor generation time.

Laboratory Safety

Treat all specimens as potentially ______________________ and pathogenic.

Personnel must wear PPE when handling patient specimens to prevent contamination of clothes and spreading pathogens to general public.

Disposable gloves are required in the microbiology lab; face masks may be needed if production of aerosol particles is likely.

Culture Media

Six types of culture media include ________________, general purpose, ______________, selective, ___________________, and enrichment.

Some media contain characteristics of more than one type.

Common laboratory media are optimized to support growth of many, but not all pathogens. Occasionally, strains of common organisms grow poorly, if at all, in the lab.

Culture Media
  • Transport media is designed to keep microbes alive while not encouraging ______________and reproduction
    • Culturette used for specimen collection contains prepared transport media
Culture Media
  • _____________________ media, or nutrient media, is not commonly used in veterinary practice.
  • _________________ media are formulated to meet the requirements of the most fastidious pathogens.
    • Basic nutrient media with extra nutrients added such as blood, serum, or egg
    • Examples: blood agar and chocolate agar
  • ____________________ mediacontain antibacterial substances such as bile salts or antimicrobials that inhibit or kill all but a few types of bacteria
    • Example: MacConkey agar
Culture Media
  • ______________________ media allow bacteria to be differentiated into groups by biochemical reactions on the media
    • Example: Simmons citrate
  • _______________________ media are liquid media that favor growth of a particular group of organisms
    • Contains nutrients that encourage growth of the desired organisms or contain inhibitory substances that suppress competitors.
    • Examples: Tetrathionate broth and selenite broth
Blood Agar
  • An enriched medium that supports the growth of most bacterial pathogens
  • Trypticase soy agar with sheep blood is most common type.
  • Blood agar acts as an enriched medium and a differential medium because four distinct types of hemolysis can be detected:
    • ___________ hemolysis – partial hemolysis that creates a narrow band of greenish or slimy discoloration around colony.
    • ___________ hemolysis – complete hemolysis that creates a clear zone around the bacterial colony
    • ___________ hemolysis – produces no change in the appearance of the medium and no hemolysis around colonies
    • ___________ hemolysis – zone of hemolysis surrounded by a narrow zone of hemolysis around a colony (aka – double-zone hemolysis)

Delta hemolysis (Double Zone Hemolysis

MacConkey Agar and EMB agar

MacConkey agar and Eosin-methylene blue agar are selective and differential media.

MacConkey agar contains crystal violet, which suppresses growth of gram-positive bacteria. Because it also contains bile salts, it is selective for bacteria that can grow in the presence of bile salts, which is similar to the environment found in the intestines.

ThioglycollateBroth (Enriched media)

Liquid medium used to culture anaerobic bacteria and determine the oxygen tolerance of microbes

Contains stable oxygen gradient, with high concentrations of oxygen near the surface and anaerobic conditions near the bottom.

Obligate aerobes will grow only in top layer; obligate anaerobes will grow only in bottom.

Facultative anaerobescan grow throughout but usually grow in middle between the zones.

Primarily used in veterinary practice as enrichment media and for blood cultures.

Other Culture Media
  • Urea tubes (Enriched media)
    • Urea slants should be streaked with inoculum and incubated overnight at 37⁰ C.
    • Urease-positive bacteria produce a pink-red color change due to hydrolysis of urea; urease-negative remains yellow.
  • Sulfide-indole motility tubes (Selective)
    • Hydrogen sulfide production is indicated by blackening of medium.
    • If positive, a red-ring forms around top of medium.
Other Culture Media
  • Simmons citrate tubes (Differential)
    • Differentiate bacteria according to use of citrate
    • Slant surface is inoculated
    • Bacterial use of citrate in medium imparts a deep blue color; unchanged medium is green.
  • Triple sugariron agar (Selective)
    • Contains an indicator system for hydrogen sulfide production and pH indicator, phenol red, which colors uninoculated medium red.
Other Culture Media
  • Bismuth sulfate agar (Selective)
    • Used when suspect salmonellae
  • Mueller-Hinton (General purpose)
    • General purpose media primarily used for the performance of the agar diffusion antimicrobial sensitivity test.
  • Sabourand dextrose and bismuth-glucose-glycine yeast media (Not for bacteria)
    • Used specifically for the culture of fungi and yeast.
    • Often an ingredient in DTM found in clinic
Combination and Modular Culture Media
  • Bullseye and Target systems
    • Five-chambered agar plates containing selective and nonselective media plus a central area with Mueller-Hinton agar for sensitivity testing.
  • “Dipslides” or “Paddle” media (Uridip® or Solarcult®)
    • Useful tools for UTI screening; made with a variety of media combinations; most common ones have either MacConkey or EMB and cystine lactose electrolyte-deficient agar.
  • Enterotubes
    • Commercially available microbiology test kits incorporating multiple types of media designed to provide differentiation of enteric bacteria based on biochemical reactions on the media.
Additional Bacterial Testing

Usually the genus of pathogenic organisms can be determined using just staining and culture characteristics (_____________________ or _______________________identification).

Please review the tables on pp. 132-133 in Lab Pro book, you will be doing this in lab!

Some organisms must be further differentiated to species level and require additional testing.

Specimen Collection
  • Aseptic technique is critical to achieving diagnostic-quality results!
  • Various methods are acceptable, including: aspiration, swabbing, scraping, depending on the type of lesion and location on animals body.
  • Samples to be processed immediately can be collected with sterile cotton swabs:
    • Contamination risk is high
    • Cotton can inhibit microbial growth
    • Oxygen can become trapped in fibers, making recovery of anaerobic bacteria less likely.
Specimen Collection

If delays in processing sample are expected, a__________ swab in transport media (Culturette) may be used to preserve quality of sample.

Specimen selected must contain organism causing the problem

Normal flora and contaminants may complicate sample collection and subsequent interpretation of results.

Better results will be obtained if specimens are collected from sites that would normally be sterile; infections are likely to be caused by a single, predominant organism.

Inoculation of Culture Media

Use aseptic technique atalltimes!

Culture plates are kept closed unless inoculating or removing colony specimens for testing.

When transferring samples to or from a tube, pass the tube neck through a flame before and after transfer of material and avoid putting the cap down.

When flaming an inoculation loop or wire, place the ____________ portion of the wire in the flame first and then work toward the contaminated __________.

Proper TechniqueStreaking of Culture Media

When the specimen collected is a liquid, a small quantity of well-mixed sample is inoculated at the edge of the plate with a sterile swab or bacteriologic loop.

Pre-sterilized glass rods may be used for streaking samples; disposable inoculating loops and wires are also available.

If the specimen has been initially collected on a sterile swab, this is streaked directly on the plate using the ____________________ method. (You will be using a swab)

A

B

D

C

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 initial area of the plate.

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.

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.

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.Step Three: (The Tertiary Streak)

Rotate the plate another 1/4 turn and sterilize your inoculating loop or take a fresh, sterile stick or swab. Again, make sure to cool your loop between streaks.

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).

You’re done! Let it grow for 18-24 hours

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.

Incubation of Cultures

For pathogens that can invade internal organs of an animal, the optimal growth temperature is usually near 37⁰ C.

For some skin pathogens (such as dermatophytes), and environmental organisms, the optimal growth temperature is lower. (Room temperature may be satisfactory)

Incubation time depends on the generation time of individual bacterial species and the type of medium on which they are growing.

Incubation of Cultures

For routine cultures, after 18 to 24 hours of incubation.

____________ culture plates during incubation so that moisture does not collect on surface of agar, which may cause clumping of colonies.

Some pathogens require carbon dioxide for growth in the culture atmosphere; a candle jar may be used.

Inoculation of Culture Media
  • If several types of colonies grow on the plate, each colony is ___________________ onto separate plates and the procedure repeated until a pure culture is obtained.
  • When using tube media, either surface of slant is inoculated or the butt and slant may be inoculated
    • Butt first (Inner portion of media toward bottom of tube.
    • “S” shaped streak on slant surface

A

B

Primary Identification of Bacteria

Systematic approach needed to identify pathogenic bacteria.

Flow charts of bacteria seen most often and the tests used to differentiate those bacteria can be used.

Specimens are first streaked onto a primary medium, such as blood agar and MacConkey agar.

Plates are incubated for 18 to 24 hours and examined for growth.

Further identify suspected pathogens on the incubated plate regarding genus and/or species with the flow chart.

Primary Identification of Bacteria

Most gram-positive and gram-negative organisms grow on_____________________.

Gram-positive organisms usually do not grow on MacConkey agar, but it can support growth of most gram-negative organisms.

Selection of the colony from the routine blood agar plate is preferable rather than from MacConkey agar.

Colony Characteristics
  • Help to identify the bacterium involved and include:
    • _____________________ (In millimeters; described as pinpoint, medium, large)
    • _____________________ (color; grey, yellow, white, creamy, black….)
    • _____________________ (opaque, transparent)
    • _____________________ (raised, flat, convex, drop-like)
    • _____________________ (circular, irregular, rhizoid, filamentous, undulate)
    • _____________________ (glassy, smooth, mucoid, buttery, brittle, sticky)
    • _____________________ (sweet, pungent, etc.)
    • _____________________ (alpha, beta, gamma, delta, none)
Staining of Microbiology Samples
  • Samples taken directly from patients are often______________ stained before being cultured.
  • Information obtained from direct smear may help determine:
    • Suitability of the specimen for identification
    • The predominant organism in a mixed specimen
    • Appropriate medium for culture
    • Appropriate antibacterials for sensitivity testing
Gram Staining Procedure

Swab specimens may be rolled lightly onto the slide.

Touching the sterile wire to one colony on the plate is usually sufficient to obtain enough bacteria for application to the slide.

Colonies should be young (24-hour culture) because older colonies may not yield proper results and the stained bacteria often become excessively _____________________ .

Gram Staining Procedure

Bacterial samples from plates are gently mixed in a drop of water or saline on the slide.

Samples may be obtained from inoculated broth by spreading two to three loops-full onto the slide.

Sample may be smeared directly onto a slide, such as from tissue or an abscess.

Sample droplet on slide may be encircled with wax pencil or Sharpie to help find area after staining.

Gram Staining Procedure
  • After the material has dried on the slide, it is heat fixed by passing the slide through a flame two or three times, specimen side up.
      • Prevents sample from washing off, helps preserve cell morphology, and kills the bacteria, rendering them permeable to stain.
  • Slide is placed on a staining rack over a sink.
  • _____________________ solution is poured onto the smear and allowed to stand for 30 seconds.
  • Slide is rinsed gently from the back with water (tap water is acceptable).
Gram Staining Procedure

_____________________ solution is poured onto the smear and allowed to stand for 30 seconds.

Slide is gently rinsed from the back with water

Smear is washed with_____________________ until no purple washes off (usually <10 seconds)

Slide is rinsed with water.

Basic fuchsinor safranin is poured on the smear and allowed to stand for 30 seconds.

Smear is rinsed again with water.

Smear is blotted dry with _____________________ paper.

Gram Staining Procedure

Smear is examined microscopically with the 100x oil-immersion objective.

Bacteria that retain the crystal violet-iodine complex and stain purple are gram _____________

Bacteria that lose the crystal violet or purple color and stain red are gram _____________.

To ensure proper staining quality, stain known (control) gram-positive and gram-negative organisms at least once per week and with each new batch of stain.

Other Microbiology Staining Procedures
  • Potassium Hydroxide (KOH) Test
    • Used when a gram-_____________ reaction occurs.
  • Acid Fast Stain
    • Used primarily to detect Mycobacterium and Nocardiaspecies.
    • Contain several solutions, including a primary stain (typically dimethylsulfoxide – DMSO and carbolfuchsin), an acid-alcohol decolorizer, and a counterstain, such as NMB.
    • After final rinse, if color remains, the organism is “acid-fast” and appears red, whereas, non-acid fast microorganisms stain blue.
Other Microbiology Staining Procedures
  • _____________: Used to detect spirochetes and rickettsiaeand to demonstrate the capsule of Bacillus anthracis.
    • Smear is fixed in absolute methanol for 3 to 5 minutes and air dried.
    • Then, smear is dipped in diluted stain for 20 – 30 minutes.
    • Bacteria stain purplish-blue.
Other Microbiology Staining Procedures
  • Specialized Stains
    • Have limited application in the average veterinary practice
    • Flagella stains
      • Usually contain crystal-violet
      • Are used to detect and characterize bacterial motility
      • Usually expensive; there are other methods of testing motility
    • Capsule stains
      • Used for detection of pathogenic bacteria
        • All bacteria that contain capsules = pathogenic
        • Not all pathogenic bacteria contain capsules
      • Requires use of bright-field phase contrast microscopy
Other Microbiology Staining Procedures
  • Endospore stains
    • Bacterial spores contain protein coats of keratin that make them resistant to most normal staining procedures.
    • Detect presence, location, and shape of spores
    • Older culture is used (>48 hours)
    • Involves addition of malachite green to specimen and counterstaining with safranin or basic fuchsin
    • Spores appear dark blue/green with the remainder of bacterial cell pink or red.
  • Fluorecent stains
    • Used primarily for identification of Legionellaand Pseudomonas
    • Expensive.
Quality Control Cultures

Monitor procedures and supplies for quality and accuracy, including antibacterial susceptibility tests, media, biochemical tests, and certain tests for identification.

A selection of control organisms can be obtained on disks.

Bacteria can be stab inoculated into a tube of medium and subcultured every ~2 months.

Quality Control Cultures

Streptococcus, Pasturella, and Actinobacillusspecies die quickly on culture plates.

Streptococci can be kept in a test tube of cooked meat broth and subcultured every ~4 weeks.

Pasturellaand Actinobacillus spp. Remain viable if mixed with approximately 0.5 ml of whole blood in a small tube and stored in a deep freeze at -10⁰ C or lower.

Control cultures can be kept at room temperature in screw-capped tubes but preferably in a refrigerator at 4⁰ C, which reduces the metabolic rate of the organisms.

Antibiotic Sensitivity Testing

Performed to determine the susceptibility or resistance to specific antimicrobial drugs

Designed for rapidlygrowingbacteria.

Specimen used for testing is taken from animal prior to beginning pharmacologic treatment

Agar diffusion method uses paper disks impregnated with antimicrobials.

Concentration of drug in disk chosen to correlate with therapeutic levels of drug in animal being treated

Most common method is Kirby-Bauer test.

Kirby-Bauer Disk DispenserAntibiotic Sensitivity Testing

__________________________are measured to determine bacterial resistance or susceptibility to specific antimicrobial drugs.

MIC = Minimum Inhibitory Concentration; this is the smallest concentration of a specific antimicrobial that can inhibit the growth of a given bacteria.

MIC can be determined using a method similar to agar diffusion test or using a broth dilution susceptibility test.

Zones of InhibitionAgar Diffusion Method
  • Antimicrobial disks are placed on the inoculated agar surface with a disk dispenser or sterile forceps that have been flamed and cooled between each use.
    • Disks should be no closer than 10 to 15 mm from edge of plate.
    • Separate disks from each other sufficiently to avoid overlapping zones of inhibition.
  • Plates are inverted and incubated aerobically at 37⁰ C and placed in the incubator within 15 minutes after placing the disks on the inoculated agar.
  • Plates are read after 18 to 24 hours
  • Prolonged incubation may alter the size of zones of inhibition or make them difficult to read.
Agar Diffusion Method

Determine antibiotic susceptibility by physical measurement of the inhibitory zones.

That measurement is compared to a chart of inhibitory zones to determine the relative resistance of the bacterium to the antibiotics being tested.

Diameter of the zone (including the disk) is measured from the underside of the plate by calipers, transparent ruler, or template and recorded to the nearest millimeter.

Agar Diffusion Method
  • Inhibitory zones are divided into two major categories: _____________ and ________________ to the particular antimicrobial agent.
  • Susceptible strains are subdivided into intermediately susceptible and susceptible.
  • Test susceptible reference organisms regularly, preferably in parallel with each batch of antimicrobial susceptibility tests.
    • Control organisms are used to check growth-supporting capability of the medium, potency of antimicrobial disks, and other variable conditions that can affect the results.
Urine Culture Colony Count

Presence of pathogenic bacteria does not necessarily indicate infection; small numbers of organisms may be found even in samples normally considered sterile like urine.

Colony count on cultured samples can help support a diagnosis of infection.

Performed by streaking a blood agar or other nonselective agar plate using a calibrated loop containing 10 microliters of urine.

After incubation, all colonies are counted and multiplied by 100 to determine the number of colony-forming units per milliliter.

Urine Colony Count
  • Significant numbers of CFUs per milliliter of urine:
    • Cystocentesis: >1,000
    • Catheter: > 10,000
    • Voided samples: >100,000 (dogs); >10,000 (cats)
Mastitis Testing

Mastitis is caused by bacterial or mycotic organisms.

Several laboratory tests diagnose mastitis, including the California mastitis test, somatic cell count, and milk culture.

Bacteria can be quickly detected by examining a thin smear of mastitic milk that has been heat fixed and stained with Gram stain or methylene blue.

CMT is a qualitative screening test that can be used as a “Cow-side test”

California Mastitis Testing

2 ml of milk is placed in each of 4 cups on the CMT paddle and an equal amount of reagent is added.

Paddle is gently rotated for ~10 sec. in a circular pattern; a score is assigned for each cup.

Test is based on gel formation when the test reagent reacts with DNA in somatic cells; as the cell count of milk increases, the gelling action increases.

Degree of gel formation scored as negative, trace, 1, 2, or 3, and y (acidic - purple) or + (alkaline - yellow)

Reaction must be scored 10 to 15 sec. after mixing starts.

California Mastitis TestMilk Culture
  • Positive milk samples identified by CMT should be cultured.
  • Milk sample inoculated on blood agar and MacConkey agar and incubated at 37⁰ C for 24 hrs
    • A tube of milk sample is also incubated simultaneously
  • If cultures show minimal or no growth after 24 hrs., a subculture is made on the plates from the incubated tube of milk.
  • Subculture is incubated for an additional 24 hrs.

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