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VET140- Microbiology
Bacterial Culturing & Staining
| Term | Definition |
|---|---|
| The technique of isolating bacteria in pure culture form was perfected by: | Robert Koch |
| Bacterial Dx includes: | -patient hx, CS's, gross observation of lesion(s), review other test results, collect specimen, direct gram stain of specimen, inoculate culture media incubate for 18-24 hours. |
| Clinical History Includes: | -signalment (species, age sex, spay/neuter, age), pertinent hx, number affected, how long have they been sick, CS's, treatments. |
| Specimen Collection Parts | -label: should note if zoonotic potential -take your time -send to lab in a timely fashion -reference lab's submission form |
| Collection Methods | -FNA, impression smear, aerobic/anaerobic culture, special media for fastidious organisms, fungal cultures, transtracheal wash, cultures of body fluids, biopsy of tissue for culture. |
| When to Collect | -collect in acute stages of dz -prior to any tx (abx) -blood cultures during febrile episodes -smears examined right away or prepared for shipping. |
| Sterile Body Areas | -blood, urine, spinal fluid, joint fluid, solid organs, lower respiratory tract. |
| Nonsterile Body Areas | -when healthy contain normal flora to be distinguished from pathogens, hair, skin, saliva, sputum, intestinal tract w/ feces, ears, upper respiratory tract. |
| Abscessed Body Areas | -filled w/ exudative material -sterile abscess = not produced by infection and often heal and scar -primary infection: 1 organism, secondary infection, multiple opportunistic pathogens. |
| Milk Collection | always discard the first few squirts when collecting this sample |
| Urine Collection | this can be collected by cysto, catheter, void-note method on form. |
| Blood Collection | this should be collected whole and be unclotted. |
| Eye Sample Collection | this sample can be collected through a conjunctival swab, lacrimal secretions, corneal scraping. |
| Bacterial Identification Steps | direct gram stain > inoculate > incubate > observe culture results > gram stain of colonies > continue with identification process (additional media and biochemical testing) > possible detection/identification by molecular methods. |
| Slide 18? | |
| 18 pt 2? | |
| Diff Quik | a rapid method to identify presence or bacteria or fungi. |
| Gram Stain | rapid method to ID specific types of bacteria based on cell wall components. |
| Ziehl-Neelsen Stain | stain used for Mycobascteria spp. |
| Modified Ziehl-Neelsen | stain used for Coxiella, Brucella, Nocaria, and Chlamydia spp. |
| Sample from Swab | this sample collection method can be directly rolled onto a slide. |
| Sample from Agar Plate | add drop of water to slide > touch wire loop to 1 bacteria colony on the plate > transfer to a slide > once bacteria is added mix bacteria w/ water and spread/smear on slide. Young colonies should be used. |
| Broth Sample Guidelines | if taking sample from an inoculated brother used 2-3 wire loopfuls on a slide, encircle sample droplet on the slide w/ a wax pencil, this will help you find the area after staining. |
| Heat Fixing | allow specimen to dry, then do this. Pass the sample through the tip of the flame 3-4 times specimen side up. Do not overheat. Prevents sample from washing off and helps preserve cell morphology, kill bacteria, and permeable enough to stain. |
| Gram Staining Procedure | pour Crystal Violet onto smear, stand for 30-60 secs, rinse, pour iodine solution, stand 30-60 secs, rinse, allow decolorizer to sit for 3-5 secs, rock slide about 10 secs, rinse and replace on rack, pour safranin, stand 30-60 secs, rinse, air dry. |
| Gram Positive Bacteria | this bacteria will retain the crystal violet-iodine complex, results in bacteria staining blue-purple-positive, forgetting to decolorize or not decolorizing enough can yield a false positive. |
| Gram Negative Bacteria | this bacteria will lost the violet stain and purple/blue color through decolorization process, take up the safranin and stain red-pink-negative. Over-decolorizing or using an old colony/weak stain can cause a false negative. |
| Gram-variable Reaction | this occurs when the stain has excess decolorization, thick smear, excessive heating, old cultures, or a poor quality stain. Can figure out if +/- by a KOH Test. |
| KOH (potassium hydroxide) Test | test used to differentiate gram-variable reactions. A gram (-) will be a sticky strand and mucoid mass. Gram (+) will have no strand and no mucoid mass formed. |
| Reasons for Heat Fixing (test question) | Prevents sample from washing off, helps preserve cell morphology, kills the bacteria, and renders them permeable to stain. |
| How to do KOH Test | place loopful of KOH on slide > place culture sample onto KOH slide > stir 30 secs > slowly lift loop and look for sticky strand/mucoid mass. |
| Solid or Semi-solid media | facilitates isolation of individual microbial colonies when there are multiple organisms. |
| Broth | used to recover small numbers of organisms or more fastidious ones. |
| Plating from a liquid specimen | plating from this type of specimen involves placing a few drops with sterile syringe or pipette on or in the media. |
| Plating from a swab | plating from this type of specimen requires applying the sample directly to the plate or the tip being placed directly in broth. |
| Plating from a tissue specimen | plating this type of sample requires: searing surface to decontaminate > flame loop placed into this sample > apply to plate. |
| Dx of Gram (-) Dz | this type of bacteria has no growth. It will need to be re-incubated, rechecked. If no growth after that, then report "no growth." |
| Dx of a Gram (+) Dz | this type of bacteria will have growth of colonies. Select representative colonies > gram stain > continue w/ ID process, possible detection/identification by immunological or molecular methods. |
| Inoculating for Identification | make sure to take care to prevent contamination, use aseptic technique, keep culture plates closed, flame tub neck before and after transfer, flame loop, do not put down lid, put sterile swab directly onto plate. |
| Streaking of Culture Plates | the preferred method of this is the quadrant method. If several types of colonies grow, then each is sub-cultured onto separate plates. |
| Quadrant Method | overlap streaked area 1-2 times between quadrants > use entire plate, keep lines close together without overlapping, place into incubator upside down. |
| Inoculation of Agar Slants | only use surface of a straight flamed wire, streak in S-shape. Butt is stabbed w/ tip of straight flamed wire, surface streaked in an S-shape, replace cap. |
| Culture Media | used to grow and identify bacteria, |
| Agar | semisolid media, dried extract of seaweed and gelatin. |
| Tube | screw-top container; contains broth or agar |
| Slants | tube of agar that allowed to gel on an angle, some are inoculated only on the slant part some into the butt. |
| Plate | flat round container of media, wire loop inoculation for isolated colonies. |
| Culture Evaluation | this should be done 12-48 hrs of incubation, look for: # of colonies, color, form, elevation and margin of each colony, hemolytic reactions on BAP, anaerobic/aerobic, describe reactions or fermentation results. |
| Identification of Bacteria (things to look for) | -is specimen sterile? -normal flora? -common pathogens for the specimen -pathogen suspected? -growth from contamination? -colony characteristics |
| Colony Growth Characteristics | -size, elevation, form or margin, density, texture, pigment, odor, hemolysis on blood sugar. |
| Spreading | bacterial colony growth either from center or edge. |
| Swarming | bacteria can glide across surfaces as they grow leaving a film over the entire plate surface, can inhibit growth of other bacteria (Ex: Proteus sp.) |
| Enriched Media (nutritive) | for initial growth of bacteria, often used for initial isolation of bacteria (some main contain blood, some egg, some serujm). |
| Selective Media | media that has compounds to inhibit growth of certain organisms, e.g., MacConkey (ihibits Gram positive). |
| Differential Media | med that has compounds that identify certain characteristics of organisms grown on meds, e.g., urea. |
| Trypticase Soy Agar | a basic media, grows about everything, easy to see colony morphology and pigment production. |
| Trypticase Soy Agar w/ sheep blood (TSA) or Blood Agar Plate (BAP) | media that supports growth of most bacteria, use for further testing vs. MacConkey, also differential because of distinct types of hemolysis that can be detected. |
| Alpha Hemolysis | a partial hemolysis that creates greenish marrow band around colony. |
| Beta Hemolysis | complete hemolysis that creates a clear halo around colony. |
| Gamma Hemolysis | no hemolysis or discoloration. |
| Hemolysis | the rupture or destruction of red blood cells. |
| Brain-Heart Infusion Broth (BHIA) | the brain and heart tissue of sheep, pre-enrichment before plated, often used for blood cultures. |
| Trypticase Soy Broth | media that grows most bacteria including aerobic, anerobic, and facultative aerobic organisms, used for blood cultures, also used for sterility testing. |
| Thioglycollate Broth (THIO) S p 244 | media used to culture anaerobic bacteria to determine the oxygen tolerance of organisms, the medium has a stable oxygen gradient-higher concentrations of oxygen at top with anaerobic conditions at the bottom, used as enrichment media for blood cultures. |
| MacConkey II Agar Plate | supports growth of Enterobacteria and other gram (-) bacteria. Crystal violate in it suppresses growth of gram positives, bile salts in it are selective for certain gram negatives. Lactose fermenting organisms = media bright pink. Poss differential media. |
| Differential Media Examples | display visible differences caused by growth specific colonies |
| Mannitol Salt Agar | selective for staphylococci, not commonly used. |
| Bismuth Sulfite Agar | selective, suppresses, coliforms and permits salmonellae. |
| Mueller-Hinton Agar | used for antimicrobial sens. |
| Sabouraud Dextrose (DTM) | media used for fungi and yeast, |
| Optimal Growth Temp | 37*C (98.6*F) |
| Incubation Time | for routine cultures incubate for 48 hrs and examine @ 18-24 hours. *Some organisms such as Nocardia species may take 72 hours or more* |
| Humidity | 70-80% |
| Ziehl-Neelsen or Acid Fast: | some bacteria have a coating that is resistant to the decolorizer used with the gram stain procedure, if you perform a gram stain on these bacteria you will see an abnormal gram positive. |
| Most Notable Acid-Fast Organisms | -Mycobacterium and Nocardia |
| Acid-Fast Stain Steps | thin smear > air dry + heat fix 2-3 times > cover slide w/ carbol-fuchin stain, heat = steam 5 mins, cool, wash, decolorize w/ an acid alcohol decolorizer, counter stain w/ TB Methylene Blue for 30 secs, wash, examine w/ 100 X oil immersion lens. |
| Dilute Carbol Fuchsin or Modified Ziehl-Neelsen | this stains bacteria red, is great for bacteria with capsules. (Ex: Campylobacter spp., Brachyspira spp., Fusobacterium spp.) |
| Polychrome methylene blue | stain that uses Polychrome methylene blue, Bacillus anthracis in blood smears (stains blue with pink capsules). |
| Diff Quik | the Romanowsky stain, can be ed with dry or heat fixed slide prep. All bacteria and fungi stain blue and epithelial cells can be blue or pink. WBCs stain varies by type. |
| Giemsa or Wright | the Romanowsky stain, primary stain for blood cytology. Stains bacteria and fungi blue. |
| Catalase Test | this tests for the enzyme catalase, commonly used to differentiate staphylococci from streptococci. |
| Catalase Test Steps | place colony sample on slide > add frop of 3% hydrogen peroxide. Gas = catalase +. No gas = catalase negative. Blood agar itself reacts and can create a positive result. |
| Staphyloccocus | a catalase positive bacteria |
| Streptoccocus | a catalase negative bacteria |
| Coagulase Test | test to see if coagulase is present. Done on catalase-positive gram positive cocci. |
| Coagulase Test Steps | place 0.5 mL of diluted lyophilized plasma in a test tube inoculate tube w/ colony, incubate tube and read hourly for 4 hrs, (-) reaction= no clot formation / (+) reaction indicated by clot formation. |
| Oxidase Test | tests for cytochrome C oxidase enzyme-oxidase positive organisms can utilize oxygen. |
| Oxidase Test Steps | few drops of oxidase test reagent to piece of filter paper > streak bacteria onto paper > (+) reaction will turn bacterial violet-purple immediately (10-30 secs). |
| Wet Prep | loopful of culture examined under coverslip |
| Hanging Drop | modified slide, petroleum jelly |
| Motility | this is determined b |
| Sulfide- Indole- Motility (SIM) Test | test done with stab agar with straight wire. Takes 24-48 hours. |
| Urea Test | test done using a streak slant with wire and incubated overnight. Pink media = (+) result (meaning the organisms hydrolyzes urea) and no color change yellow = (-) |
| Triple Sugar Iron (TSI) Test | test that uses sugar fermentation and checks for hydrogen sulfide production. |
| Yellow | this color indicates A (acidic) on a Triple Sugar Iron Test. |
| Red | this color indicates K (alkaline) on a Triple Sugar Iron Test. |
| K/K | this indicates red slant and red butt: none of sugars fermented in Triple Sugar Iron Test |
| K/A | indicates red slant and yellow butt: glucose fermentation only in the Triple Sugar Iron Test. |
| A/A | indicates yellow slant and butt: fermented glucose, lactose, and sucrose in the Triple Sugar Iron Test. |
| Blackening of medium | this indicated hydrogen sulfide production in the Triple Sugar Iron Test. Read before black reaches bottom of tube. |
| Simmons Citrate Tubes | this tells whether an organism utilizes citrate, streak slant only, deep blue color media is a positive result if the bacteria use the citrate, no color change (green) is a negative results |
| Antimicrobial Susceptibility Testing | this test determines the susceptibility or resistance of bacteria to antimicrobial drugs |
| Antimicrobial Susceptibility Testing Steps | take sample from >1 colony > inoculate broth, strea Mueller-Hinton agar, apply abx discs 20-24 mm apart. press discs, incubate within 1 mins, incubate 18-24 hrs, keep refrigerated until used. |
| Inhibitory Zone | this must be measured sring antimicrobial suscepibility testing. May read after 6-8 hrs if rapid results are neccessary and then confrim at 18-24 hrs, underside of plate, diameter of the zone including the disc is measured, record to nearest mm. |
| Results: Susceptible | this result indicated no growth of bacteria around disc. |
| Results: Resistant | this result indicated bacteria grow up to disc. |
| Minimum Inhibitory Concentration Test (MIC) | the smallest concentration of a specific antimicrobial that can inhibit growth of bacteria. Disks of different concentrations of drugs used. |
| Microbial Drug Resistance (slide97) | a major problem in human medicine and becoming significant in vet med. Widespread + indiscriminate use of abx produces resistant strains of bacteria that become predominant bacterial populations . |
| Factors affecting in vivo activity of abx | -site and rate of absorb -site of excretion -tissue distribution -metabolism -interactions between pathogen and drug -interactions between host and pathogen |
| Resistance | this trait can be transferred genetically to non-resistant bacteria which then acquire this. |
| Cross-resistance | resistance to other drugs within the same class. |
| Multiple-drug resistance | (mediated by plasmid) resistance to drugs from different classes. |
| Bacterial Conjugation | 2 bacteria fusing together to exchange information. |
| Bacterial Transformation | picking up plasmids from the environment |
| Bacterial Transduction | virus relocates bacterial DNA. |
| Measures control Drug Resistance | -monitor abx use/supply -data -strict adherence to drug dosages, schedules, withdrawal times. |
| Colony Count Procedure | measure water/urine in a calibrated loop, streak onto blood agar plate, after incubation all colonies are counted & times by 100 to determine # of colony-forming units per mL of sample. |
| Mastitis Tests | gel formation when test reagent reacts with DNA in somatic cells. As the cell count of the milk increases, the gelling action increases. Most common is California Mastitis Test (C MT). |
| Milk Culture | only positive CMT samples are cultured, MacConkey and blood agar are both inoculated, milk sample is also incubated so tests can be repeated if no or minimal growth occurs. |
| Acid-Fast Organisms | organisms that will show stained red. |
| Non Acid-Fast Organisms | organisms tat will show stained blue. |
| Coagulase | an enzyme that coagulates plasma. |
Created by:
Riley.Scherf
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