Presentation is loading. Please wait.

Presentation is loading. Please wait.

Microbiology Clinical Pathology.

Similar presentations


Presentation on theme: "Microbiology Clinical Pathology."— Presentation transcript:

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

8

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.

15

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 -

38

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

50

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

70

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


Download ppt "Microbiology Clinical Pathology."

Similar presentations


Ads by Google