Culture Media Flashcards
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.
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)
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)
Selective Media
Selects for growth of a group of organisms by adding inhibitory substances such as antimicrobials, dyes or alcohol.
These substances inhibit the growth of other organisms. (eg, MacConkey agar selects for growth of most facultative gram-negative bacilli and inhibits the growth of gram-positive cocci and bacilli and gram-negative cocci.
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.
Atmospheric Conditions for incubation:
Ambient (air): 21 % oxygen, 1% CO2
CO2 -enhanced: 18% oxygen, 5-10% CO2
Microaerophilic: 5%-6% oxygen, 5-10% CO2
Anaerobic atmosphere: <1% oxygen, 5- 10% CO2
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
