OverviewYou will be using a wide variety of media and biochemical tests to isolate and identify your unknown organisms. Your text has a good section on enrichment, selective, and differential media. Please review it before getting started. Show
You are only working with a small subset of the available media and tests. It is enough however to give you a good idea of the problem solving that goes on behind the scenes to support patient care. The information provided on these pages was derived from the DIFCO Manual of media, which is also available in the lab. Read the directions carefully. You must pay close attention to the conditions of the tests and media to avoid erroneous results. Things to watch for include:
Bacitracin/SXT SensitivityIntended UseBacitracin differential disks are used to presumptively identify Group A, beta-hemolytic streptococci from other beta-hemolytic streptococci. The combination of SXT sensitivity increases the accuracy of the results. PrincipleBacitracin is an antibiotic isolated from Bacillus subtilis. It inhibits cell wall synthesis mainly through inhibiting the biosynthesis of peptidoglycan. SXT inhibits folate metabolism which interferes with bacterial DNA synthesis. Group A, beta-hemolytic streptococci are more sensitive to bacitracin than other beta-hemolytic streptococci. Test ProcedureThe standard protocol has been modified for our lab.
Results
This table is from MacFaddin, Biochemical Tests for Identification of Medical Bacteria.
Limitations
CatalaseIntended Use
PrincipleHydrogen peroxide (H2O2) is the end product of aerobic breakdown of sugars. Since it is toxic to bacterial cells, most aerobic bacteria produce catalase or peroxidase to protect themselves. Streptococcus, Enterococcus, and Lactobacillis are exceptions. Since they do not use the cytochrome c pathway, they do not produce H2O2 and lack catalase. Test Procedure
Results
Limitations
Coagulase TestIntended UseDifferentiates Staphylococcus aureus from other Staphylococcus species. PrincipleThe coagulase test detects the presence of free and bound staphylcoagulase. This enzyme is excreted extracellularly by human strains of Staph. aureus. The mechanism of action is unknown. Test Procedure
ResultsThe formation of a clot in the bottom of the tube is considered a positive result. The clot will not move as you tilt the tube. Unclotted plasma will flow in the tube. Limitations
Eosin Methylene Blue (EMB) Agar Eosin Methylene Blue (EMB) AgarPrincipleA differential plating medium for the detection & isolation of the gram-negative enteric bacteria. Purpose
Test Procedure
Interpretations
Fluid Thioglycolate Medium (FTM)Tests the oxygen requirements of different microorganisms. Intended UseCharacterizes microbes according to their oxygen requirements
PrincipleVarious types of bacteria require various oxygen (or oxygen-free) environments to grow in. Test Procedure
Results
Hemolysis (Blood) Agar Hemolysis - Blood AgarIntended UseBlood agar is used to support the growth of fastidious organisms and to determine the type of hemolysis (destruction of red blood cell walls) an organism produces. PrincipleBlood agar is a rich medium that has been supplemented with fresh 5-10% blood. The hemolytic response can be dependent upon the type of blood. Sheep blood is commonly used, but some organisms require rabbit or bovine blood. Test Procedure
ResultsYou can differentiate four types of hemolysis by the appearance of the agar.
Limitations
MacConkey (Mac) Agar MacConkey (Mac) AgarPrinciple
Purpose
Test Procedure
Interpretation
Motility Test MediumIntended Use
PrincipleMotility is apparent by the presence of diffuse growth away from the line of inoculation. Test Procedure
Results
Limitations
Mannitol Salt Agar (MSA)PrincipleSelective and differential media used for the isolation of Staphylococcus aureus. Intended UseDifferentiates Staphylococcus aureus (+) from other Staphylococcus species. Principle
Test Procedure
ResultsThe yellowing of the red/pink media indicates a positive result. NovobiocinIntended UseNovobiocin Differentiation Disks are useful for presumptively distinguishing Staphylococcus saprophyticus from other coagulase-negative staphylococci (CoNS) in clinical specimens. PrincipleNovobiocin Differentiation Disks are prepared by impregnating 5µg of novobiocin onto high quality 6mm diameter filter paper disks. Novobiocin Differentiation Disks are useful in presumptively distinguishing S. saprophyticus from other CoNS. Novobiocin inhibits the synthesis of DNA and RNA. A zone of inhibition is produced by contact with the novobiocin. Procedure
Interpretation of Results
Limitations
Oxidase Biochemical AssayTests for the presence of the enzyme indophenol oxidase. Intended UseThe oxidase test is based on the production of an enzyme called indophenols oxidase. This enzyme oxidizes a redox dye (present in the reagent) which results in a color change of yellow to dark purple. PrincipleIndophenol oxidase, in the presence of atmospheric oxygen, oxidizes the phenylenediamine oxidase reagent to form a dark purple compound, indophenol. Test Procedure
ResultsPositive reactions turn the bacteria violet to purple immediately, or up to 30 seconds. Negative reactions remain colorless or turn light pink/light purple after 30 seconds. Delayed reactions should be ignored. Limitations
Salt Tolerance BrothIntended UseSalt tolerance broth is intended to differentiate non-beta-hemolytic strains of streptococci. Principle of UseBrain Heart Infusion (BHI) broth is supplemented with 6.5% sodium chloride and bromcresol purple as a pH indicator. The indicator is included to make reading the test results easier. The broth also includes dextrose. The fermentation of dextrose (glucose) results in the production of acid. This changes the pH of the media causing the media to turn from purple to yellow. Test Procedure
ResultsA positive reaction is indicated by obvious turbidity in the media with or without a color change. A negative result is indicated by no growth after 72 hours. Enterococcus spp. typically changes the media color within 24 hours. Limitations
Simmons Citrate Agar Slant Simmons Citrate Agar SlantPrincipleUsed for the differentiation and identification of Enterobacteriaceae on the basis of citrate utilization, citrate being the sole carbon source. PurposeColonies capable of utilizing citrate as a carbon source produce a local increase in pH, changing the color of the medium from green to blue. Only citrate positive organisms will grow on this medium. Test Procedure
InterpretationsGood growth with the medium color turning blue indicative of Enterobacter aerogenes and Salmonella choleraesuis. Which media contains ingredients which inhibit the growth of other organism?Selective media contain ingredients that inhibit the growth of some organisms but allow others to grow. For example, mannitol salt agar contains a high concentration of sodium chloride that inhibits the growth of most organisms but permits staphylococci to grow.
What type of media encourages the growth of certain types of organisms while inhibiting the growth of others?Media that inhibit the growth of unwanted microorganisms and support the growth of the organism of interest by supplying nutrients and reducing competition are called selective media. An example of a selective medium is MacConkey agar.
Which type of medium supports the growth?Basal Media:
This type of media supports the growth of microbes that don't need the enrichment media to form colonies. Such media are beneficial for primary isolation of the bacteria such as the fastidious microorganisms. Some of the favoured basal media are nutrient broth, peptone water, and nutrient agar.
Which medium is used to inhibit the growth of Gram positive organisms?MacConkey agar is an example of a medium that is both differential and selective. The presence of bile salts, as well as crystal violet, within the media prevent gram-positive organisms from growing.
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