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
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
Mycelium is a web of hyphaeDermatophytes
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
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
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 rodsBacterial 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” patternBacterial 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
TerminalBacterial 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
Delta hemolysis (Double Zone HemolysisMacConkey 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
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
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)
CStep One: (The Primary Streak)
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 hoursStep 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
BPrimary 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
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
_____________________ 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
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
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
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
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