Diagnostic and Biochemical Tests for Gram Positive Cocci/ Gram Negative Bacilli By: Angelita A. Briñas, RMT I. CATALASE TEST + result vigorous effervescence

II. COAGULASE TEST

III. MANNITOL SALT FERMENTATION TEST

IV.DNASE TEST * * Staphylococus aureus on the left is negative for DNase production; the Serratia marcescens on the right is positive for DNase production as evidenced by the area of clearing around the growth.

IV. NOVOBIOCIN TEST
Rapid, automated identification of novobiocin-resistant, coagulase-negative staphylococci. (CoNS)
ID of Staph.saprophyticus
Resistant= less than 16mm
Sensitive= more than 16mm * Staph.saprophyticus is Novobiocin resistant * Staph.epidermidis- Novobiocin sensitive

V.BACITRACIN (TAXO A) TEST
Difference of the group A beta-hemolytic streptococcus from other non-group A beta-hemolytic streptococci is by using sensitivity test to bacitracin (Taxo A discs).

OPTOCHIN (TAXO P) DISCS TEST
This is a differential test used to distinguish between organisms sensitive to the antibiotic optochin and those not. This test is used to distinguish Streptococcus pneumoniae (optochin sensitive (pictured on the right)) from other a-hemolytic streptococci (optochin resistant (Streptococcus mitis is pictured on the left).

Bile- Esculin Hydrolysis
Determine the ability to grow in 40% bile and esculin hydrolysis
POSITIVE RESULT- Esculetin reacts with FeCl3 to form brown-black ppt

The differential ingredient is esculin. If an organism can hydrolyze esculin in the presence of bile, the product esculetin is formed. Esculetin reacts with ferric citrate (in the medium), forming a phenolic iron complex which turns the entire slant dark brown to black. The tube on the far right was inoculated with E. faecalis (positive). The tube in the center was inoculated with a bilie esculin negative organism and the tube on the left was uninoculated.

The tube on the far right was inoculated with E. faecalis (positive). The tube in the center was inoculated with a bilie esculin negative organism and the tube on the left was uninoculated.

NOVOBIOCIn
Rapid, automated identification of novobiocin-resistant, coagulase-negative staphylococci. (CoNS)
ID of Staph.saprophyticus Resistant= less than 16mm
Sensitive= more than 16mm

Associated with bacterial Endocarditis following the insertion of artificial heart valves. Staphylococcus saprophyticus- Resistant Staphylococcus epidermidis – Sensitive

Hardy Diagnostics PYR Test Kit and PYR Reagent

Enterococcus faecalis (ATCC® 29212) growth was applied to a PYR Disk moistened with deionized water and incubated at room temperature for two minutes.
Subsequently, one drop of Chromogenic Solution (PYR Reagent) was applied to the disk. The pink to red color development was indicative of a positive PYR reaction.

CAMP Test
Streptococcus agalactiae, a member of the Lancefield Group B streptococci, is one of the causative agents of mastitis in cows.
Identifying this organism can be difficult, and the CAMP Test was designed to aid in the identification of this organism. This test relies on the fact that most S. agalactiae strains produce a diffusible, extracellular compound that will, in conjunction with a specific beta-hemolysin of Staphylococcus aureus, cause complete lysis of sheep red blood cells in an agar medium. This test was named after the authors of the original paper.

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Reverse CAMP test
Comparison of CAMP test with Streptococcus agalactiae and reverse CAMP test.
The reverse CAMP reaction is a reaction whereby hemolysis by the beta-hemolysin of staphylococci is inhibited through the production of phospholipase C or D by some organisms (e.g., some Corynebacteria). An arrow of no hemolysis is formed at the junction of the organism being tested with the staphylococci if the reverse CAMP test is positive.

Hippurate hydrolysis
The presence of the enzyme hippurate hydrolase is visualized by the purple color formed when ninhydrin reagent oxidizes the amino acid produced during hippuratehydrolysis.

The hydrolysis of sodium hippurate is a qualitative test useful in the identification of: * Streptococcus agalactiae (group B β-hemolytic streptococci), * Campylobacter jejuni, * Gardnerella vaginalis, * Listeria sp. and * other aerobic bacteria.1-4
Specimen Collection: This product is not intended for primary isolation of patient specimens. This product is used in conjunction with other biochemical tests to identify cultures of isolated organism.

Streptococcus pneumonia Viridans streptococci Both are :Not Lancefield grouped alpha hemolytic on BAP
Viridans streptococci has the ff.species: Strep.mutans- related w/dental carries Strep.mitis S. intermidius Strep. salivarius Strep. constellatus Strep. uberis
Neufeld Quellung reaction
The swelling of the capsule of a bacterium, seen in the laboratory when the organism is exposed to specific antisera. This phenomenon is used to identify the genera, species, or subspecies of the bacteria causing a disease, including Haemophilus influenzae, Neisseria meningitidis, and many kinds of Streptococci
Difference between S.pneumoniae and Strep. Viridans | Strep. pneumoniae | Viridans strep. | Mouse virulence | ( + ) RIP | (-) | Inulin fermentation | (+) | (-) | Bile solubility | (+) | (-) | Optochin(Taxo P) | Sensitive | Resistant | Neufeld Quellung | (+) capsular swelling | (-) |

BIOCHEMICAL TEST FOR ENTEROBACTERIACEAE:

IMViC Test- INDOLE TEST

Product detected: Indole
Positive result- red ring (or dark pink color)
Negative result- no color development * Variable – Orange color- indicates production of Skatole- a methylated intermediate maybe a precursor of indole production.

Sulfur Indole Motility Media (SIM)

2nd tube -Indole (+)

METHYL-RED TEST
Culture medium: MRVP broth
Reagent: Methyl red ph indicatorPositive: * Product detected: Mixed acids * Positive: Red color
Quality control: Escherichia coli (+)- red ring Klebsiella pneumonia – (-)

Voges-Proskauer test
The Voges-Proskauer test detects the presence of acetoin, a precursor of 2,3 butanediol. If the culture is positive for acetoin, it will turn “brownish-red to pink” (tube on the left) If the culture is negative for acetoin, it will turn “brownish-green to yellow” (tube on the right

An aliquot of the MR/VP culture is removed and a-naphthol and KOH are added. They are shaken together vigorously and set aside for about one hour until the results can be read.

SIMMON CITRATE UTILIZATION TEST

LYSINE IRON AGAR (LIA) Test
Culture medium : LIA – purple butt/slant * Product detected : Cadaverine * Interpretation: Lysine decarboxylation (+) = purple deep Lysine decarboxylation (-) = yellow deep Lysine deamination (+) = red slant H2S (+) = blackening * Note: purple butt-= means alkaline

* Positive decarboxylation (butt), negative deamination (slant) * Tube 2: Negative decarboxylation (butt), positive deamination (slant)

Urease test
This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease.
It is commonly used to distinguish the genus Proteus from other enteric bacteria. The hydrolysis of urea forms the weak base, ammonia, as one of its products. This weak base raises the pH of the media above 8.4 and the pH indicator, phenol red, turns from yellow to pink.
Proteus mirabilis is a rapid hydrolyzer of urea (center tube pictured here).
The tube on the far right was inoculated with a urease negative organism and the tube on the far left was uninoculated.

TRIPLE SUGAR IRON AGAR (TSI)

1.Bacteria -that cannot ferment glucose produces no change in the indicator resulting to an alka line slant and alkaline butt (K/K) 1st tube from R-L
2. Sodium thiosulfate , the sulfur source , provides sulfur atoms to detect the production of H2S gas, reacting with iron salts to produce black ppt of ferrous sulfide.
These bacteria produce an alk slant/ acid butt w/H2S (K/A)H2S+
2nd tube ( R-L)

3. Bacteria - that ferment glucose but not lactose and sucrose produce small quantity of acids that cannot counteract the degradation of amino acid in the slant , resulting in an alkaline pH due to oxidative phosphorylation. These bacteria produce an alk slant/ acid butt w/gas (K/A)G(+) (3rd tube R- L)

4.Bacteria - that ferment both glucose and lactose and/or sucrose that produce large amount of acid, which overcome the alkaline reaction in slant yielding an acid slant and acid deep.(A/A)G * (yellow slant/yellow butt/cracks)4th tube

DECARBOXYLASE REACTION

* Determines the decarboxylase production of bacteria by the use of amino acids such as Lysine, Ornithine, and Arginine * Decarboxylase – enzyme that removes the carboxyl group of a specific amino acid.
Principle-: the amino acid to be tested is incorporated to the basal medium in a 1% concentration. Each decarboxylase reaction is specific for a particular amino acid. * Culture medium: Moeller decarboxylase broths containing : Lysine (1%) Ornithine (1%) Arginine (1%) Sterile Mineral Oil * A control tube of Moeller decarboxylase medium containing glucose but no amino acid should be set up with each culture. Bromcresol purple is the pH indicator which turns purple in alkaline condition and yellow in acid.

Ortho-Nitrophenyl beta- D- Galactopyranoside (ONPG)

* Determines the presence of beta –galactosidase- enzyme produced by late lactose fermenters (LLF)
Useful in identifying late lactose fermenters

Rapid Lactose Fermenters- has lactose permease and beta Galactosidase * Late Lactose Fermenters- has beta galactosidase only * Non-Lactose Fermenter- lacks both enzyme

Principle:- ONPG structure molecule is structurally similar to lactose . It can enter the bacterial cell without permease. In the presence of beta galactosidase, ONPG is converted into galactose and o-nitro-phenyl, which is yellow chromogen and the alkaline end product, respectively. * Interpretation: Positive result = yellow color within 20 mins–24hours
Negative result= colorless after 24 hours * Quality control: Escherichia coli – (+) yellow Salmonella typhimurium (-) = no color change

* Negative- no color change after 24 hrs * Positive- yellow color after 24 hrs