Common Media and Biochemical Tests Objectives

Describe the importance of plate reading to the bacterial culture examination process.

• Characteristics and form of bacterial colonies

• Compare with direct examination

• Distinctive patterns can distinguish some pathogens and facilitate presumptive identifications

Blood Agar

Composition: Tryptic Soy Agar base with 5% sheep blood. TSA is a nutritive media that provides casein and soy peptones for source of nitrogen and amino acids

Clinical Utility: Grows fastidious organisms and can help differentiate them by hemolytics

MacConkey agar

Composition: crystal violet dye, bile salts, lactose, and neutral red (pH indicator)

Clinical Utility: isolate and identify gram-negative bacteria

Sorbitol MacConkey (SMAC)

Composition: Peptone 20.0; sodium chloride 5.0; bile salts No. 3 1.5; sorbitol 10.0; crystal violet 0.001; neutral red 0.03; agar-agar 15.0.

Clinical Utility: used to identify and isolate Escherichia coli 0157:h7 (EHEC)

Chocolate Media

Composition: added hemoglobin and a supplementary growth factor (e.g., KoEnzyme Enrichment).

Clinical Utility: isolation and cultivation of fastidious bacteria, particularly Haemophilus influenzae, Brucella Spp., and Neisseria species

Modified Thayer Martin (MTM)

Composition: hemoglobin, which provides the X factor (hemin), and GCHI Enrichment, which provides the V factor, vitamins, amino acids, coenzymes, and dextrose. Vancomycin and colistin are selective agents that inhibit gram-positive cocci and gram- negative bacilli, respectively

Clinical Utility: isolation of pathogenic Neisseria from specimens containing mixed flora of bacteria and fungi.

Hektoen Enteric (HE) Agar

Composition: Peptone base with bile salts, lactose, ferric ammonium citrate, bromthymol blue, and acid fushion

Clinical Utility: Able to isolate & differentiate Salmonella (black from hs2) & Shigella (Blue) from other enterics

Xylose Lysine Deoxycholate (XLD) agar

Composition: Similar to HE agar but contains different xylose as carbohydrate source and lysine for nitrogen

Clinical Utility: Salmonella sp will turn black, Shigella sp will be colorless (lack of xylose ferm), Any remaining non-pathogens will be orange (ferm of xylose)

Campy Blood Agar

Composition: Agar base, sheep blood, vancomycin, polymyxin b, trimethoprim, nystatin

Clinical Utility: primary isolation of. Campylobacter jejuni from stool specimens

Colistin-nalidixic acid (CNA) agar

Composition: an agar base with nutrients (peptones, starch, sodium chloride), the selective and differential antibiotics colistin and nalidixic acid, and typically 5% sheep blood

Clinical Utility: isolation and identification of gram-positives, inhibits growth of gram-negatives

Anaerobic blood agar

Composition: a basal agar with peptones, yeast extract, and agar as a base, supplemented with sheep blood for growth factors and hemolysis observation, and specific anaerobic growth stimulants like Hemin and Vitamin K

Clinical Utility: Isolation and cultivation of anaerobic bacteria

Lim/Group B Selective broths

Composition: Takes a streptococcal supportive media and adds colistin, nalidixic acid, and yeast extract

Clinical Utility: Selectively grows Group B Streptococci from the material Genitourinary tract to avoid neonatal sepsis, any growth seen can be subculture to BAP to determine if Group B strep is present

Staphylococci

Streptococci

Diptheriods

Enterics

Yeast

small, visible clusters of cells that are often creamy-white to pinkish in color, with a smooth, moist, or slightly rough surface texture

Catalase

Purpose of test:Detects if the organism can break down hydrogen peroxide into water and carbon dioxide

clinical utility: differentiates Staph (+) and Strep (-)

Reagents: Hydrogen Peroxide

Limitations: do not use colonies from blood containing agar since rbcs pseudoperoxidase activity of heme

Coagulase

Purpose of test: detects if the organism can convert soluble fibrinogen into insoluble fibrin in a plasma sample

Clinical Utility: Differentiates Staph aureues (+) from couagulase negative staph (-)

Reagents: Fibrin-containing plasma

Limitations: Other organisms besides s.aureus can be positive

Oxidase

Purpose of test: Detects the presence of a cytochrome oxidase that will catalyze the transport of electrons between electron donors in the bacteria

Clinical Utility: Aids in the differentiation of Neisseria, Moraxella, Campylobacter, and Pasteurella species

Reagents:methyl-p-phenylenediamine dihydrochloride, in water

Limitations:

(O/F) Tests

Purpose of test: If the organisms utilize carbohydrates aerobically or anaerobically

Clinical Utility: understanding organism metabolism

Reagents:

Limitations:

TSI and KIA slants

Purpose of test: Aims to determine a microorganism’s ability to ferment various sugars, produce H2S, and/or produce gas

Clinical Utility: Used to differentiate enterics from other GNB’s and also differentiate among the enterics

Reagents:Agar contains peptones, 1% Lactose, 1% sucrose, and 0.1% glucose along with sodium/ferrous thiosulfate (reagent in media), and phenol red

Limitations:

Methyl Red/Voges-Proskauer

Purpose of test: MR: Detects acid end product detection from ferm of glucose VP: Test is used to determine if an organism produces acetylmethyl carbinol from glucose fermentation.

Clinical Utility: Historically used to differentiate enterics, Now used to differentiate Actinobacteria

Reagents: Both: media containing peptones and glucose

MR detection uses methyl red pH detector

VP detection uses alpha-napthol/KOH detector combo

Limitations: Can not be both MR and VP pos

Phenylalanie deaminase (PAD)

Purpose of test: To determine the ability of an organism to oxidatively deaminate phenylalanine to phenylpyruvic acid.

Clinical Utility: Used to differentiate Proteus, Providencia, and Morganella orgs

Reagents: Agar contains nutrients and phenylalanine After incubation, 10% Ferric Chloride - if positive will turn black

Limitations:

Pyrrolidonyl arylamidase (PYR)

Purpose of test: pyrolidonyl arylamidase activity in Streptococcus spp, Enterococcus spp., some coagulase-negative staphylococci, and some Enterobacteriaceae

Clinical Utility: identify group A and D strep (+)

Reagents: N, N-dimethylaminocinnamaldehyde to look for red color (+)

Limitations:

Bile-Esculin

Purpose of test: Can the organism hydrolyze esculin in the presence of bile

Clinical Utility: Used to differentiate Streptococci bovis - negative and Enterococci - positive

Reagents: Nutritive agar with esculin and bile with ferric salt

Limitations:

Salt Tolerence

Purpose of Test: Can the organism survive and grow in a 6.5% salinity?

Clinical Utility: Used to differentiate enterococci from Group D strep and Aerococcus spp 

Reagents: Brain-heart infusion broth (nutritive media) with 6.5% NaCl solution

Limitations:

Amino Acid decarboxylase

Purpose of test: Determines what amino acids an organism can decarboxylate. Mainly use arginine, ornithine, and lysine as the amino acid

Clinical Utility: Differentiate decarboxylase producing Enterobacteriaceae from other GNB’s.

Reagents: The agar media contains nutrients, the amino acid being assessed, and a pH indicator. Decarboxylation raises pH causing purple color (+)

Limitations:

Lysine Iron Agar (LIA)

Purpose of the Test: Determines if an organism can decarboxylate or deaminate lysine and if it can form H2S

Clinical Utility: Used to differentiate enterics and other GNB’s

Reagents: lysine, peptones, a small amount of glucose, ferric ammonium citrate, and sodium thiosulfate.

Limitations

OF Sugars

Purpose of the Test: Determines if an organism oxidizes or ferments sugars

Clinical Utility: Differentiate various organisms

Reagents: Agar-based stab media with nutrients and the sugar of interest with pH indicator

Limitations

Indole

Purpose of Test: Determines if an organism can decompose tryptophan to indole

Clinical Utility:Used to differentiate various enterics

Reagents:organism is incubated in a tryptophan broth and allowed to incubate then Kovac’s reagent (p-Dimethylaminobenzaldehyde) is added to determine if pink color (+) forms

Limitations

Hippurate Hydrolysis

Purpose of Test: Determines if an organism can hydrolyze Hippuric acid into glycine and benzoic acid

Clinical Utility: Used to identify Group B strep, Campylobacter jejuni, Listeria monocytogenes, Gardnerella vaginalis

Reagents: Prepare a heavy inoculum of isolated organism to a test tube and add disk with Hippurate into it. After 2 hour incubation add nihydrin reagent and look for purple color change to occur (+)

Limitations:

Nitrate Reduction

Purpose of Test: Determines if organism reduces nitrate as terminal electron acceptor in anaerobic metabolism

Clinical Utility: to differentiate various enterics by other by products being produced

Reagents: Broth contains nutrients and nitrate.Sulfanic acid is added after incubation to detect nitrites and then alpha-naphthylamine to give a red precipitate

Limitations: When negative must add zinc powder to reduce any lingering nitrate. If pink after zinc: negative. If not pink after zinc: all nitrate was already used and thus a positive test

H2S Production

Purpose of Test: Determines if an organism can reduce sulfur into H2S

Clinical Utility: Differentiates various enterics

Reagents: Nutrient-rich media with Sodium thiosulfate.

Limitations

Citrate Utilization

Purpose of Test: Determines if an organism can use citrate as a sole source of energy - use as carbon source

Clinical Utility: Differenitiate varius enterics

Reagents: Agar contains no carbohydrates and no carbon sources besides sodium citrate. Bromothymol blue pH indicator is present. When citrate is broken down (energy used), ammonium salts that are formed and converted to ammonia. The alkaline pH shift causes color change from green to blue (+)

Limitations

Urease

Purpose of Test: Determines if an organism can produces the enzyme urease - lowers Ph

Clinical Utility: Enterics

Reagents: Agar contains no carbohydrates and no carbon sources besides Urea. Phenol red pH indicator is present. When urea is broken down, ammonium salts that are formed and converted to ammonia. The alkaline pH shift causes color change from yellow to pink (+)

Limitations: Prolonged incubation= false positive

Butyrate Esterase

Purpose of test: detect enzyme butyrate esteraste

Clinical Utility: Identify Moraxella Catarrhalis

Reagents: Pad impregnated with bromo-chloro-indolyl butyrate has isolated colony placed onto it. Hydrolysis releases indoxyl which oxidizes and forms a violet color

Limitations

Bile Solubilty

Purpose of test: detect if the organism autolysis occurs when exposed to bile salts

Clininical Utility: Identify Strep Pneumo

Reagents: Prepare a suspension of organism and then add sodium deoxycholate

Examine for clearing of turbidity after 15 minutes

Limitations

The principle and utility of hybridization-based detection systems in medical microbiology

Principle: The formation of hydrogen bonds between single strands of RNA or DNA that are complementary - Target - Sequence that will be identified - Probe - Single-stranded RNA or DNA oligonucleotide labeled with a reporter chemical, radionucleotide, or fluorescent particle.

Utility: Deteermines recognition of the target sequence at the 5’ or 3’ end

The principle and utility of PCR detection systems in medical microbiology

PCR: Denaturing, Primer Annealing, Primer Extension

The principle and utility of MALDI-TOF technology

Useful for identifying microorganisms in clinical microbiology laboratories

Identification can occur in minutes from isolated colonies

They are separated by their mass-to-charge ratio.