01- (TCEPP) Introductory Microbiology and Susceptibility Testing (Exam #1) Flashcards
Prokaryote
Organisms without a true nucleus
Size: 0.5-5 µm
(Bacteria)
Eukaryote
Organisms with a true nucleus
Size: >10 µm
(Fungi, Parasites)
Facultative Anaerobes
Grow equally well aerobically and anaerobically
Aerobic
A condition in which microorganisms grow with oxygen.
Anaerobic
A condition in which microorganisms grow without oxygen
Enteric pathogen
Microorganisms in the gastrointestinal tract that cause disease.
Normal flora
Microorganisms that normally live in various areas of the human body.
Facultative anaerobe
Microorganisms that grow equally well aerobically and anaerobically
BT (Bioterrorism)
The deliberate release of viruses, bacteria or other germs (agents) used to cause illness or death in people, animals, or plants.
Nosocomial Infection
An infection acquired in a health care setting
Flagella
An anatomical feature that allows most bacteria to become motile.
Monotrichous
Bacteria that have a single flagellum (e.g., Vibrio cholerae)
Lophotrichous
Bacteria that have multiple flagella located at the same spot on the bacterial surfaces which act in concert to drive the bacteria in a single direction. (e.g., H. pylori, Pseudomonas putida)
Amphitrichous
Bacteria that have a single flagellum on each of two opposite ends (only one flagellum operates at a time, allowing the bacterium to reverse course rapidly by switching which flagellum is active). (e.g., Campylobacter jejuni)
Peritrichous
Bacteria that have flagella projecting in all directions (e.g., E. coli)
What type of stain is the Gram Stain?
-A Differential stain placing most bacteria into 2 groups- gram-positive and gram-negative.
Explain the Heat Fixation Method
To heat fix, you can pass the slide quickly through a flame 2 to 3 times or by placing the slide on a slide warmer at 65ºC for 10 minutes. Allow the slide to cool to room temperature.
Explain the Methanol Fixation Method
- Methanol fixation can be performed by applying methanol to the air dried slide for 1 minute, draining off excess by tilting the slide, then allowing it to completely air-dry
* Methanol fixation is the preferred method, as it does not create aerosols and has fewer changes in cellular morphology.
Important Reminders about fixation of slides
- Allow the smear to air dry completely before fixing with heat or methanol.
- Heat fixing before the smear has completely air dried can cause aerosolization and/or cell breakage, causing the cells to appear distorted after staining.
- Methanol fixing the smear before it is completely dry, can cause the specimen to wash off the slide.
- Excessive heating can crack the microscope slide and/or rupture the cells of the specimen.
- Fixing the smear kills the organisms and causes the cells to adhere to the microscope slide so they do not wash off during the staining process
Differential Stain
A staining technique that allows the distinct visual difference between organism structures or components. For example, Gram positive bacteria stain dark purple and Gram negative bacteria stain red to pink.
Explain the difference between Gram-Positive and Gram-Negative Bacteria
- Due to the difference in the cell wall structure of Gram-positive and Gram-negative bacteria, they will retain the Gram stain differently.
- Gram-positive bacteria have cell walls with a thick layer of peptidoglycan, allowing them to retain the primary stain. The primary stain, crystal violet, will cause Gram-positive bacteria to stain purple in color.
- In contrast, Gram-negative bacteria have cell walls with a thin layer of peptidoglycan and high lipid content. These bacteria stain pink because crystal violet is not retained through the decolorization step.
Explain The Gram Staining Process
- Apply the primary stain, crystal violet, to a fixed smear
- Add mordant, Gram’s Iodine
- Decolorize primary stain using decolorizer
- Counterstain using safranin
Describe
Primary Stain (step #1)
- The first reagent of the Gram stain is crystal violet. It is an alkaline dye and the first stain applied to a fixed smear.
- During this step, the crystal violet dye is binding to the cell wall of the bacteria.
- If you look at the slide under a microscope at this point, the smear will be purple and all cells will be stained purple.
Describe
Mordant (step #2)
- Gram’s iodine (depending on the kit, this may be called by a different name) is the second reagent used when performing a Gram stain. This is known as the mordant.
- During this step, the iodine binds with the crystal violet to form a crystal violet-iodine, insoluble, complex that binds to the peptidoglycan layer of the bacterial cells.
- After this step, if you look at the slide under a microscope, the smear and all cells will still be purple.
Describe
Decolorizer (step # 3)
- Decolorizer is the third reagent used in the Gram stain procedure. An acetone–alcohol mixture or pure alcohol can be used to perform this step.
- During this step, the decolorizer removes the lipid membrane from Gram negative cells. This results in leaching of the crystal violet–iodine complex, leaving the Gram-negative cells colorless. Gram-positive cells will retain the primary stain; remaining purple in color.
- This step is the most crucial because cells can easily become under–decolorized, if the decolorizer is not left on long enough; or over–decolorized if the decolorizer is left on too long.
- At this stage the smear may appear colorless, but under a microscope the Gram-positive cells will be purple and the Gram-negative cells will be colorless.
Describe
Counterstain (Step # 4)
- The fourth and final reagent of the Gram stain is safranin. Safranin is used as a counterstain.
- During this step, the counterstain will stain the colorless cells of Gram-negative bacteria, pink. If you do not add the counterstain, the Gram-negative cells will remain colorless.
- Looking at this slide under a microscope, the Gram-positive cells will be dark purple and the Gram-negative cells will be red to pink in color.
Describe
Results and Interpretation of Gram Stain
- Gram-positive cells will appear dark purple, after the Gram stain procedure, due to retaining the crystal violet–iodine complex within the thick peptidoglycan layer.
- Gram-negative cells will appear red to pink, after the Gram stain procedure, due to the crystal violet–iodine complex leaching out during the decolorization process; then being counterstained with safranin.
What are some reasons for Gram Variable Results?
Some bacteria can produce Gram variable results, in which there will be both Gram-positive and negative cells.
Gram variable outcomes may also be a result of:
- An uneven direct smear
- A direct smear made from an older culture
- Damaged cell walls
- Under–decolorization
- Over–decolorization
What are some Common Problems with the Gram Stain?
- The quality of your smear preparation will directly affect the quality of your Gram stain. For example, smears that are too thick can lead to erroneous Gram stain results because the cells are clumped together.
- Under decolorizing your smear can cause Gram-negative cells to retain the crystal violet-iodine complex. This will cause the cells to appear purple instead of pink.
- Over decolorization can occur if you leave the decolorizer on the slide too long. This will result in Gram-positive bacteria losing the crystal violet–iodine complex, therefore staining pink as seen in the picture to the right.
When is the gram stain performed?
It is routinely performed on certain specimen types, especially those collected from normally sterile sites.
Why is the gram stain important or helpful?
Gram stain morphology can guide species identification
What type of organisms have a thick peptidoglycan layer in their cell wall and retain the purple crystal violet/iodine complex during the decolorizer step?
Gram positive organisms
What type of organisms have a thin peptidoglycan cell wall with lipopolysaccaharide outer membranes and lose the crystal violet/iodine complex during the decolorization step and are visualized using the red safranin counterstain?
Gram negative organisms
EXAMPLES OF ARRANGEMENT OF BACTERIAL CELLS
GRAM-POSITIVE BACTERIA
MORPHOLOGY OF BACTERIAL CELLS
(SOME BASIC SHAPES)
Gram stain morphology
Staphylococcus aureus
Gram positive cocci in clusters (grape-like)
Gram stain morphology
Micrococcus spp.
Large Gram-positive cocci in clusters and tight packets of tetrads
Gram stain morphology
Enterococcus faecalis
Gram-positive cocci, often oval form diplococci and short chains
Gram stain morphology
Streptococcus pneumoniae
- Gram-positive cocci in pairs
- Lancet forms with pointed ends
Gram stain morphology
Aerococcus spp.
Gram-positive cocci in singles and clusters
Gram stain morphology
Corynebacterium spp.
- Gram positive bacilli, “diphtheroid-like”
- Often arranged in V forms or pallisades
- Chinese letter appearing
Gram stain morphology
Bacillus spp
- GPB with blunt ends (boxy)
- Tendancy to form long chains, w/ or w/o spores, can be GVB
Gram stain morphology
Clostridium perfringens
- Straight anaerobic gram positive bacilli with blunt ends.
- Able to form capsules
- Spores are rarely seen (subterminal)
Gram stain morphology
Listeria monocytogenes
Small gram positive rods often coccobacilli
Gram stain morphology
Lactobacillus spp.
Straight gram positive rods, often in chains
Gram stain morphology
Streptococcus pyogenes
Gram positive cocci in chains
Gram stain morphology
Neisseria species
Gram negative cocci that typically appear in pairs with opposing sides flattened “kidney bean or hamburger bun” appearance
Gram stain morphology
Moraxella catarrhalis
Gram negative diplococci (kidney bean/hambuger bun)
Gram stain morphology
Escherichia coli
Gram negative bacilli
Gram stain morphology
Klebsiella pneumoniae
- Gram-negative bacilli (short, chunky, blunt ends)
- Has the ability to form capsules
Gram stain morphology
Pseudomonas aeruginosa
Gram-negative bacilli (thin, small)
Gram stain morphology
Acinetobacter baumannii complex
Gram-negative coccobacilli (plump)
Gram stain morphology
Pasteurella multocida
Tiny Gram-negative coccobacilli, bipolar staining may be present
Gram stain morphology
Haemophilus influenzae
Gram-negative bacilli / coccobacilli pleomorphic, strand forms
SMALL
Gram stain morphology
Campylobacter jejuni
**Small Gram-negative spiral-shaped, sea gulls
Gram stain morphology
Yersinia pestis
- Gram negative short rods or coccobacilli
- Tend to retain staining at the end of the cells (bipolar staining)
- “Safety pin” appearance
Quantitative Isolation or Streaking
Primarily used for urine cultures. Plates are inoculated using a calibrated loop to deliver a specified volume. The urine is mixed well, and the calibrated loop (0.01 or 0.001 mL) is vertically inserted into the urine and transferred to the culture medium by making a single streak down the center of the plate. Without flaming, the loop is streaked back and forth through the original inoculum.
The number of colonies that grow are multiplied by the dilution factor. (eg, if 0.001mL loop is used, 35 colonies would translate into 35,000 colony forming units. (CFU/mL)
Semi-quantitative or Isolation Streaking
The general-purpose isolation streak is useful for most specimens. The relative number of organisms can be estimated based on the extent of growth beyond the original area of inoculum. Growth in the first quadrant can be graded as 1 +, or light growth; growth in the second or third quadrant can be graded as 2 + to 3 +, or moderate growth; and growth in the third or fourth quadrant can be graded as 4 +, or heavy growth. Also Rare, Few, Moderate or Many Growth.
Nonselective media
Adequately supports the growth of most microorganism. (eg, trypticase soy agar)
Enriched media
Growth enhancers such as 5% sheep blood or vitamins are added to nonselective media (eg, 5% sheep blood agar, chocolate agar)
Differential media
•Employs factor(s) which allows colonies to demonstrate metabolic characteristics to distinguish them from others
Example: MacConkey agar (MAC) which differentiate gram-negative bacilli that can ferment lactose from those that do not ferment lactose.
Some media can have characteristics that would allow them to be classified as enriched AND differential or selective AND differential.
Blood Agar
(BAP)
Primary Media
Enriched (Sometimes considered differential due to hemolytic reactions.)
• Included for most specimens
• Almost all bacteria & yeast will grow
• Sheep blood is useful for distinguishing species that hemolyze the red blood cells around and under the colony
Ingredients: Trypticase soy agar; 5% sheep blood
Chocolate Agar
(CHOC)
Primary Media
Enriched
• Included for most specimens
• Almost all bacteria & yeast will grow, including fastidious species such as Haemophilus spp, Neisseria gonorrheae and Francisella tularensis
Ingredients: GC agar base with meat and casein peptones; 2% hemoglobin provides X factor (hemin); IsoVitaleX provides V factor (NAD)
MacConkey Agar
(MAC)
Primary Media
Selective & Differential
• Included for most specimens that may contain Enterobacteriaceae or mixtures of flora
• Gram-negative rods will grow
• Gram-positive organisms are inhibited
• Lactose-fermenters are pink; non-lactose fermenters are clear colonies
Ingredients: Peptone base; bile salts & crystal violet inhibit gram-positive organisms;1% lactose (sole carbohydrate source); neutral red indicator is pink-red if lactose is fermented
Campy Blood Agar (CAMPY)
Stool Culture Media
Enriched & Selective
• Isolation of Campylobacter species in stool cultures
• Antibiotics inhibit gram-positive and gram-negative flora organisms in stool
• Cultures are incubated at 42°C in microaerophilic conditions to help inhibit normal enteric (stool) flora & allow Campylobacter to grow
Ingredients: Brucella agar base with sheep blood provide heme and other growth factors; five antimicrobial agents: trimethoprim, vancomycin, amphotericin B, polymyxin, cephalothin.
Campy Filter Agar Plate
(Stool Culture Media)
Physical selection on enriched media
• Organisms are very narrow and motile, allowing passage through pores in the filter; other enteric organisms found in stool are retained on top of filter
• Cultures are incubated at 42°C in microaerophilic conditions to help inhibit normal enteric (stool) flora & allow Campylobacter to grow
Ingredients: Sheep blood agar (BAP) with 0.45 micron filter
Hektoen Enteric Agar (HEK)
(Stool culture media)
Selective & Differential
• Differentiates enteric pathogens from enteric flora in stool cultures
• Salmonella spp. produce green colonies with black centers; Shigella spp. produce green colonies
• Lactose-fermenters e.g. E. coli produce orange colonies; non-lactose fermenters are green or green with black centers
• Gram-positive organisms are inhibited
Ingredients: Yeast extract & peptone base; bile salts inhibit gram-positives; lactose, sucrose, salicin differentiate fermenters and non-fermenters; ferric ammonium citrate & sodium thiosulfate determine H2S production (black center in colony); bromthymol blue indicator
MacConkey Broth
(Stool culture media)
Enriched & Selective
• Used for isolation of Enterobacteriaceae, especially shiga toxin-producing E. coli.
• Tubes should be incubated with caps loosened during incubation to allow air exchange.
• Organisms that ferment lactose e.g. E. coli turn the media yellow and cloudy.
Ingredients: Peptone base; bile salts (Oxgall) inhibit gram-positive organisms; lactose; Brom Cresol purple indicator
MacConkey Sorbitol Agar (MACSORB)
(Stool Culture Media)
Selective & Differential
• Isolation and differentiation of sorbitol-negative E. coli such as some Shiga toxin-producing strains (e.g. E. coli O157:H7)
• Sorbitol-negative colonies are clear; sorbitol positive colonies are pink
• Gram-positive organisms are inhibited
Ingredients: Peptone base; bile salts & crystal violet gram-positive organisms; 1% sorbitol (sole carbohydrate source); neutral red indicator
Selenite F Broth
(Stool Culture Media)
Enriched & Selective
• Added to enhance isolation of Salmonella & Shigella; at CCF used with pediatric samples
• Selenite inhibits Enterococcus and Enterobacteriaceae that are part of the normal gut flora
• After 12 to 18 hrs incubation the broth is sub-cultured to an agar plate for isolation of Salmonella & Shigella
Ingredients: Pancreatic digest of casein (peptones); cysteine; sodium phosphate; lactose; sodium selenite
Thiosulfate Citrate Bile Salts Sucrose (TCBS)
(Stool Culture Media)
Selective & Differential
• Isolation of Vibrio spp. from stool specimen
• Salt content inhibits most other gram-positive & gram-negative organisms
• Yellow colonies = V. cholerae or V. alginolyticus
• Green colonies = V. parahaemolyticus
Ingredients: Yeast extract & peptone base; sucrose is fermentable carbohydrate; Oxgall; sodium cholate, sodium chloride, bile salt; sodium thiosulfate; sodium citrate; bromthymol & thymol blue pH indicator
Yersinia Agar (CIN) Cefsulodin-Irgasin-Novobiocin Agar
(Stool culture media)
Selective & Differential
• Isolation of Yersinia spp. & sometimes Aeromonas spp. in stool cultures
• Yersinia is a pink colony
• Antibiotics inhibit most stool flora
Cultures are incubated at room temperature.
Ingredients: Yeast extract & peptone base; bile salts & crystal violet inhibit gram-positive organisms; mannitol; cefsulodin, irgasin & novobiocin inhibit normal stool flora; neutral red indicator
Xylose-Lysine-Deoxycholate Agar (XLD)
(Stool culture media)
• Used to isolated Salmonella spp. and Shigella spp.
• Incubated at 35°C for 24 hrs in ambient air, four different colony morphologies are possible.
o Yellow
o Yellow with black centers
o Red
o Red with black centers
Ingredients: Sucrose, lactose and xylose with phenol red being the color indicator.
Potato Dextrose Agar (PDA)
(Mycology culture media)
Enriched
• Clinically important yeast and fungi will grow.
• Primarily for sterile sites since no inhibitory antibiotics are added
• Stimulates production conidia which are used in microscopic identification; stimulates pigment production from dermatophytes
Ingredients: Potato starch infusion and dextrose base; tartaric acid is added to lower pH, inhibiting bacterial growth
Potato Dextrose Agar with Chloramphenicol (PDACH)
(Mycology culture media)
Enriched & Selective
• Added to specimens containing bacterial flora such as respiratory, skin, and wound sites.
• Addition of chloramphenicol to PDA helps inhibit bacteria
Ingredients: Potato starch infusion and dextrose base; tartaric acid is added to lower pH, chloramphenicol inhibits bacterial growth
Candida CHROMagar
(Mycology culture media)
Differential & Selective • Selective medium for the isolation and presumptive identification of yeast. o Candida albicans/dublinensis – green o C. tropicalis – metallic blue o C. krusei – pink o Other species - white to mauve
Ingredients: Peptone and agar base with 0.5% chloramphenicol for bacterial inhibition. A proprietary chromogenic mix is added for color differentiation of Candida spp.
Mycosel/Mycobiotic Agar (MYCO)
(Mycology Culture Media)
Enriched & Selective
• Added to contaminated specimens such as skin, hair, and nails.
• Dermatophytes and dimorphic molds will grow
• Cycloheximide inhibits some pathogenic fungi such as Cryptococcus neoformans, Aspergillus fumigatus, some Candida, and most zygomycetes.
Ingredients: Peptone and dextrose base; cyclohexamide inhibits saprobic fungi & chloramphenicol inhibits bacteria
Lowenstein Jensen (LJ)
(Mycobacteriology Culture Media)
Enriched
• Used for isolation of Nocardia because media provides lipids required for growth
• Most commonly used for isolation of Mycobacteria
• Malachite green provides minimal selectivity, preventing some bacterial growth
Ingredients: Potato flour, egg, glycerol, asparagine, potassium phosphates, magnesium sulfate, sodium citrate, malachite green inhibits bacterial flora
7H11
(Mycobacteriology Culture Media)
Enriched
• Used for isolation of Mycobacteria. Can be used for growth of Nocardia
• Non-selective
• Can be purchased as a slant or plate
Ingredients: Chemically defined base with OADC enrichment (oleic acid, albumin, dextrose, catalase); contains a small amount of malachite green
7H11 Selective
(Mycobacteriology Culture Media)
Enriched & Selective
• Used for isolation of Mycobacteria, especially from specimens likely to be contaminated with bacterial flora.
• Contains antibiotics & an antifungal to inhibit growth of most gram-positive and gram-negative bacteria, yeast, and mold
• Can be purchased as a slant or plate
Ingredients: 7H11 medium with OADC; trimethoprim, amphotericin, carbenicillin & polymixin B; contains a small amount of malachite green
Bacteroides Bile Esculin Agar
(BBE)
Selective & Differential
• Used for anaerobic cultures
• Isolation and identification of Bacteroides fragilis group
• Used for specimens likely to contain mixed anaerobic flora
• Esculin is hydrolyzed yielding esculetin which reacts with ferric ammonium citrate to form a brown-black coloration
• Cultures are incubated in an anaerobic environment
Ingredients: Nutritive base of casein and soybean peptones; 20% bile inhibits gram-positives and most anaerobes other than B. fragilis group; gentamicin inhibits most facultative anaerobes; vitamin K; esculin & ferric ammonium citrate produce brown color if esculin is hydrolyzed
BCSA Agar (Burkholderia cepacia Selective Agar)
Selective & Differential
• Selects for Burkholderia cepacia in respiratory specimens, especially from cystic fibrosis patients
• Inhibits most other respiratory flora
• B. cepacia colonies & surrounding media will be yellow
Ingredients: Casein peptone & yeast extract base; crystal violet, gentamicin, vancomycin & polymix B inhibit most respiratory flora; sucrose & lactose; phenol red indicator
Buffered Charcoal Yeast Extract Agar (BCYE)
Enriched
• Isolation of Legionella spp.
• Also useful for isolation of Francisella and Nocardia
Ingredients: Nutritive base is yeast extract; α–ketoglutrate, iron, & L-cysteine are specific nutrients required by Legionella; charcoal is a detoxifying agent and surface tension modifier
Buffered Charcoal Yeast Extract Agar with PAC or PAV (antibiotics) (BCYE-sel)
Enriched & Selective
• Isolation of Legionella species from specimens contaminated with respiratory flora
• Antibiotics inhibit normal respiratory flora: gram-positive & gram-negative organisms and yeast
Ingredients: BCYE agar with antibiotics: either PAC (polymyxin B, anisomycin & cefamandole) or PAV (polymixin B, anisomycin & vancomycin)
CDC Anaerobic Blood Agar (CDC)
Enriched (Sometimes considered differential due to hemolytic reactions on blood)
• Primary medium used for cultivation of anaerobic bacteria
• Non-selective, gram-positive & gram-negative rods and cocci will grow.
• Supports obligate anaerobes, facultative anaerobes, microaerophiles & aerobes
• Cultures are incubated in an anaerobic environment
Ingredients: Trypticase soy agar with added yeast extract and 5% sheep blood; vitamin K1; hemin; cysteine
Colistin Nalidixic Acid Agar or Columbia CNA Agar
Selective (sometimes considered differential)
• Included if gram-positive organisms need to be separated from gram-negative
• Gram-positive aerobic organisms such as Streptococcus, Enterococcus, & Staphylococcus will grow
• Gram-negative organisms are inhibited
• Hemolytic reactions will be observed
Ingredients: Columbia agar base with 5% sheep blood; colistin & nalidixic acid inhibit gram-negative organisms; blood allows the detection of hemolytic reactions and provides X factor (heme).
Eosin-Methylene Blue Agar (EMB)
Selective & Differential
• Inhibits Gram-positive bacteria
• E. coli can ferment lactose and will form blue-black metallic colonies where-as Enterobacter spp. will form pink colonies. Nonfermenter colonies are colorless or light purple.
Ingredients: Eosin Y and methylene blue dyes, selective ingredients to inhibit Gram-positive organisms. Lactose and sucrose are incorporated for fermentation.
Kanamycin Vancomycin Laked Blood Agar (KVLB)
Selective
• Supports the growth of Bacteroides spp. and Prevotella spp., yeast and kanamycin resistant facultative gram negative organisms will also grow.
Ingredients: Kanamycin will inhibit most facultative gram-negative bacilli, vancomycin which inhibits most gram-positive organisms. Laked blood accelerates the brown-black colony color produced by some Prevotella spp.
Martin Lewis Agar (ML)
Enriched & Selective
• Selects for pathogenic Neisseria including N. meningitidis & N. gonorrhoeae.
• Most gram-positive and gram-negative organisms and yeast are inhibited
• Included for all genital specimens
Ingredients: Chocolate agar base; high concentration of vancomycin inhibits gram-positive; anisomycin inhibits yeast; colistin inhibits gram negative rods; trimethoprim inhibits swarming Proteus
Modified Thayer Martin Agar (MTM)
Enriched & Selective
• Selects for pathogenic Neisseria including N. meningitidis & N. gonorrhoeae.
• Most gram-positive and gram-negative organisms and yeast are inhibited
• Used for genital specimens
Ingredients: Similar to ML agar; incorporates nystatin instead of anisomycin
Mannitol Salt Agar (MSA)
Selective & Differential
• Useful in the recovery and ID of Staphylococci spp. from mixed microbiota.
• S. aureus can ferment mannitol (yellow) whereas other Staphylococcus species (pink) can tolerate the salt content but not ferment the mannitol.
Ingredients: High salt concentration (7.5%), mannitol, phenol red being the color indicator.
Phenylethyl Alcohol Agar
(PEA)
Selective
• Included if gram-positive organisms need to be separated from gram-negative; included for anaerobic cultures
• Gram-positive aerobic and anaerobic organisms such as Streptococcus, Enterococcus, Staphylococcus, & Peptostreptococcus will grow
• Facultative gram-negative rods such as Enterobacteriaceae are inhibited
• Some anaerobic gram-negative rods will grow.
Ingredients: Nutrient agar base with sheep blood; phenylethyl alcohol inhibits facultative anaerobic gram-negative bacilli; hemolytic reactions are less reliable on PEA than other blood-containing media
Spectra MRSA Agar
Selective & Differential
• Selects for methicillin resistant Staphylococcus aureus (MRSA)
• MRSA is most often screened from nares, axilla, umbilicus, rectal & perianal sites, but other sites may be screened by culture
• Colonies are blue
Ingredient: Content of media is proprietary, but will contain oxacillin or another selective antibiotic
LIM Broth
Enriched & Selective
• Used for selective enrichment of Group B Streptococcus (S. agalactiae) from vaginal-rectal swab specimens
• After 18-24 hr incubation, turbid broth is subcultured to an agar plate for isolation of Group B Streptococcus.
Ingredients: Todd Hewitt broth base containing dextrose peptones, yeast extract & salt
Nalidixic acid & colistin suppress gram-negative bacteria.
Thioglycolate Broth (THIO)
Enriched
• Detects a wide range of bacteria, including many anaerobes
• Helps to recover organisms in low numbers, especially in normally sterile body sites
• To prevent oxygenation media should not be shaken
• If >30% is oxidized (pink) media may be heated once in boiling water and cooled prior to use. (image left)
• When cloudy the broth is subcultured to solid agar for growth & identification of organisms
Ingredients: Dextrose, peptone, L-cystine & yeast extract base; thioglycolate consumes oxygen, permitting growth of anaerobes; cystine is a reducing agent, low concentration of agar assists in redox maintenance; resazurin is an oxidation-reduction indicator, being pink when oxidized & colorless when reduced.
Trypticase Soy Broth (TSB)
Enriched
• General purpose broth used for culture
• Broth used for sterility cultures
Ingredients: Enzymatic digest of casein and soybean meal, salts, dextrose
Gradient diffusion test (Epsilometer test “Etest”)
MIC is the point where the zone of inhibition intersects the strip
The strip has a gradient of antibiotics on it, place on bacterial lawn (0.5 McFarland), measure
Microbroth dilution method (<=0.1mL broth volume)
Quantitative
Indicates concentration of drug necessary to inhibit or kill the microorganisms tested
Doubling dilutions of antibiotic in broth
Turbidity visualization = MIC
50µl or 100 µl is directly dispensed into each of the 96 wells of the microtiter tray containing lyophilized antibacterial agents
The trays are examined for growth using a reflected viewing apparatus. The growth from broth alone is used as a comparison (positive control) .
The MIC is the lowest concentration that inhibits the visual growth of the organism
Zone of Inhibition
Zone related to disk diffusion testing; a clear area surrounding an antimicrobial disk following overnight incubation; results from diffusion of the antimicrobial molecules into the agar and inhibition of growth of the test bacterium.
Bactericidal
Antimicrobial that kills a microorganism.
Or amount of antimicrobial agent required to kill.
Read any amount of growth in a microdilution MIC
For E-Test see picture
Bacteriostatic
Antimicrobial that inhibits bacterial growth but does not kill the bacteria.
Measured as ≥80% reduction in growth of the organism as compared to the control well.
For E-Test see attached picture
Example: Trimethoprim/sulfamethoxazole (SXT), Linezolid
Macrobroth dilution
(>=1 ml broth volume) – Very rarely performed in clinical labs.
Agar Dilution MIC
A series of plates containing various concentrations of each antimicrobial agent are prepared.
Test bacteria (0.5 McFarland) are spot-inoculated onto each plate using a multipronged replicating device.
After overnight incubation, the MIC is read as the lowest concentration of antimicrobial agent that inhibits the visible growth of the test bacterium
Currently considered the reference method for antimicrobial susceptibility testing of anaerobes and Neisseria gonorrhoeae.
Vitek 2 (for Automated Antimicrobial Susceptibility Testing)
This system facilitates standardized susceptibility testing in a closed environment with validated results and recognition of an organism’s microbial resistance mechanism in 6 to 8 hours for most clinically relevant bacteria.
Inoculum is automatically introduced via a filling tube into a miniaturized plastic 64-well, closed card containing specified concentrations (low, mid and high) of antibiotics.
Cards are incubated in a temperature-controlled incubator
Optical readings are performed every 15 minutes to measure the amount of light transmitted through each well and compared to the growth well without antibiotics.
Algorithmic analysis of the growth kinetics in each well is performed by the system’s software to derive the MIC data
The MIC results are validated with the Advanced Expert System (AES) software.
Disk Diffusion Method: (Kirby Bauer)
Allows categorization of most bacterial isolates as susceptible, intermediate or resistant to a variety of antimicrobial agents. (Qualitative)
The antibiotic diffuses from the disk, gradually decreasing in concentration as the distance from the disk increases.
At a critical point the amount of antibiotic is unable to visibly inhibit the organism being tested; thus, a zone of inhibition develops.
The point of this zone of inhibition is directly related to the MIC value.
Disk Diffusion Method: (Kirby Bauer) Reading and Interpretation
The diameter of each inhibition zone is measured using a ruler or calipers. Plates are placed a few inches above a black, nonreflecting surface, and zones are examined from the back side (agar side) of the plate illuminated with reflected light.
Disk Diffusion results can be correlated to the MIC
The qualitative results of disk diffusion assay (KB) correlates well with quantitative results from MIC tests
Name and Recognize
Antimicrobial Targets and Mechanisms of Action (6)
1. Inhibition of Bacterial Cell Wall Biosynthesis
2. Inhibition of Folate Synthesis
3. Interference with DNA Replication
4. Interference with DNA Transcription
5. Interference of mRNA Translation
6. Combined Mechanisms of Action
Diagram where the β-lactamase enzyme cleaves the penicillin structure.
