BLOOD Blood culture is used to diagnose bacteremia, a condition

where bacteria are present in the blood. Venous blood is drawn aseptically and inoculated to

blood culture bottles. The bottle is then incubated at 36 C ̊ for 1 week and aliquots are subcultured every 1, 3, 5 & 7 days on agar plates.

If a typical pathogen is found in blood culture it is almost always significant

A contamination which mean a nonsignificant finding is probable: CONS (coagulase negative Staph), diphtheroids, Bacillus sp.

CSF CSF is collected to diagnose bacterial meningitis Major pathogens: Strep. pneumoniae, H. influenzae type

b (Hib), and Neisseria meninigitidis. Lumbar spinal puncture CSF should be collected before administration of

antibiotics, usually during the acute stage of illness to avoid the loss of viability of the etiologic agent.

Gram stain and serology BAP, CAP, MAC, BHI: BAP and CAP incubated for 72 hours

at 35-36˚C in 5-10% CO2 atmosphere

URINE Calibrated inoculating loop (0.001 mL or 0.01 mL) A colony count of 105 CFU/mL is criterion used to

determine if the organism identification and sentivitty testing are to be performed.

If the bacteria recovered are CONS additional test should be performed to identify S. saprophyticus

Neisseria N. meningitidis can be identified using Kovac’s oxidase

test and carbohydrate utilization. If the oxidase test is positive, carbohydrate utilization

testing should be performed. If the carbohydrate utilization test indicates that the

isolate may be N. meningitidis, serological tests to identify the serogroup should be performed.

Agglutination reaction Agglutination occurs when the antisera bind to the bacterial cells causing the cells to agglutinate or clump together, thus making the

cell suspension appear clearer. The intensity of the agglutination reaction may vary according to the density of the cell suspension or the antisera used.

4+ All of the cells agglutinate and the cell suspension appears clear 3+ 75% of the cells agglutinate and the cell suspension remains slightly cloudy 2+ 50% of the cells agglutinate and the cell suspension remains slightly cloudy 1+ 25% of the cells agglutinate and the cell suspension remains slightly cloudy +/- Less than 25% of the cells agglutinate and a fine granular matter occurs 0 No visible agglutination; the suspension remains cloudy and smooth

Catalase Test

The catalase test is used primarily in differentiation between certain genera and species of bacteria. Catalase is an enzyme present in most cytochrome containing aerobic and facultative anaerobic bacteria. An important exception is

Streptococcus species. The test is performed by exposing the test organism to hydrogen peroxide and observing for oxygen production. Because red blood cells contain catalase and will give a false-positive result, the test cannot be performed if blood agar is introduced. Colonies older than 18-24 hours may lose their catalase activity, and produce a false-negative result. Hydrogen peroxide is unstable and breaks down easily on exposure to light. The major test reaction to use in Staphylococcus identification is the coagulase test reaction, which divides the genus Staphylococcus

into 2 groups—coagulase negative species and coagulase positive species. The enzyme coagulase, produced by a few of the Staphylococcus species, is a key feature of pathogenic Staph. The enzyme produces

coagulation of blood, allowing the organism to "wall " its infection off from the host's protective mechanisms rather effectively.

Optochin test S. pneumoniae strains are sensitive to the chemical optochin (ethylhydrocupreine hydrochloride). Optochin sensitivity allows for the

presumptive identification of alpha-hemolytic streptococci as S. pneumoniae, although some pneumococcal strains are optochin-resistant. Other alpha-hemolytic streptococcal species are optochin-resistant.

Bile solubility test The bile (sodium deoxycholate) solubility test distinguishes S. pneumoniae from all other alpha-hemolytic streptococci. S. pneumoniae

is bile soluble whereas all other alpha-hemolytic streptococci are bile resistant. Sodium deoxycholate (2% in water) will lyse the pneumococcal cell wall.

Quellung reaction For proper quellung-based serotyping, a high quality microscope is required. A positive quellung or Neufeld reaction is the result of the

binding of the capsular polysaccharide of pneumococci with type specific antibody contained in the typing antiserum. Pneumococcal typing sera are commercially available as pooled, group, or serotype-specific

An antigen-antibody reaction causes a change in the refractive index of the capsule so that it appears “swollen” and more visible. After the addition of a counter stain (methylene blue), the pneumococcal cells stain dark blue and are surrounded by a sharply

demarcated halo which represents the outer edge of the capsule.

Biochemical Testing for Enterics TSI: tests for 3 things---sugar fermentation (glucose/lactose/sucrose), CO2, and H2S. The outcome of sugar use is always acid, so the pH indicator phenol red will turn yellow---reported as A. No use of the sugar or alkaline

by-products (which is NO sugar use) from the other non-sugar nutrients in the medium will cause the indicator to stay the same color red/orange or maybe even change it to a red (if peptones/proteins are used as an energy source, producing alkaline products)---reported as a K.

A/A = glucose and lactose and/or sucrose are used K/A = glucose alone K/K = no sugars used

Lysine Iron agar is used in the qualitative determination of lysine decarboxylation, lysine deamination, and hydrogen sulfide production. The medium can only be used with organisms that are capable of glucose fermentation. Lysine Iron agar contains dextrose as fermentable carbohydrate, lysine as an amino acid, bromcresol purple as a pH indicator, and

ferric ammonium citrate and sodium thiosulfate as sulfur source and hydrogen sulfide indicator. Initially the organism ferments glucose, causing a production of acid and changing of the pH indicator in the butt to yellow. If an organism produces decarboxylase enzymes, the organism will decarboxylate lysine to produce cadaverine, an alkaline product.

The production of cadaverine will cause the pH indicator to change back to purple. If the organism is able to deaminate lysine, the amine converts to alpha-ketocarboxylic acid and the slant turns red. If the organism is not able to deaminate or decarboxylate the lysine the butt will remain yellow, and the slant will remain purple.

Lysine decarboxylation (detected in the butt) Positive test – purple slant/purple butt (alkaline) K/K Negative test – purple slant/yellow butt (acid) K/A (fermentation of glucose only) Lysine deamination (detected in the slant) Positive test – red slant Negative slant – no color change (slant remains purple) Hydrogen sulfide production Positive test – blackening of medium (black precipitate) Negative test – no blackening of the medium

SIM (SULFIDE-INDOLE-MOTILITY) Principle 1. To determine the ability of an organism to liberate

hydrogen sulfide (H2S) from sulphur bearing amino acids producing a visible, black colour reaction.

2. To determine the ability of an organism to split indole from the tryptophan molecule.

3. To determine if the organism is motile or non-motile. The amino acid tryptophan can be converted by the

enzyme tryptophanase into an end product called indole. This chemical is identified when it reacts with Kovac's reagent.

Sodium thiosulfate is in this medium and can be utilized by bacteria, with the production of hydrogen sulfide. H2S is colorless. Ferric ammonium sulfite is in the medium to reaction with H2S, producing a black ferrous suflide.

MRVP tests are run together in the same broth and then split into 2 tubes when ready to be tested for the end products.

The methyl red test determines the use of glucose, with the subsequent production of acid, tested for by the pH indicator methyl red.

The Voges- Proskauer test also determines glucose use, but for a different end product---not acid but a neutral product called acetoin (or acetylmethylcarbinol).

Simmon's Citrate agar slants contain sodium citrate (the only carbon source) and ammonium ions (the sole nitrogen source). A pH indicator, Bromothymol Blue is also included. Bromothymol Blue is GREEN at pH < 7.0 and BLUE at pH > 7.6.

Organisms that utilize citrate for energy produce alkaline compounds as by-products. Thus, a positive result for citrate utilization is the formation of a BLUE color.

Results + = Positive (BLUE agar) - = Negative (Green agar with little to no evidence of

growth)

Urease test This test is used to differentiate organisms based on their

ability to hydrolyze urea with the enzyme urease.

Urinary tract pathogens from the genus Proteus may be distinguished from other enteric bacteria by their rapid urease activity .

Urea can be hydrolyzed to ammonia and carbon dioxide by bacteria containing the enzyme urease.

Many enteric bacteria (and a few others) possess the ability to metabolize urea, but only members of Proteus, Morganella, and Providencia are considered rapid urease-positive organisms.

It contains urea, peptone, potassium phosphate, glucose, and phenol red. Peptone and glucose provide essential nutrients for a broad range of bacteria.

Potassium phosphate is a mild buffer used to resist alkalinization of the medium from peptone metabolism. Phenol red, which is yellow or orange below pH 8.4 and red or pink above, is included as an indicator.