Bacteriology; Baileys and Scott (PARTIII_Sec.01: Chpt_12): Overview of Bacterial Identification Methods and Strategies

Microscopic identification of bacteria and its clinical specimen, Macroscopic identification of colony and its morphology, Additional microscopy for organism category identification

These are the steps in the entire workup of bacteria identification

Microscopic identification of bacteria and its clinical specimen

This step in the bacteria workup process involves providing the clinician with immediate therapy for life-threatening infections before further organism identification

Macroscopic identification of colony and its morphology

This step in the bacteria workup process involves identifying the type of hemolysis, pigment, size, texture, adherence to agar, pitting in agar, and other characteristics

Additional microscopy or gram stain observation

This step in the bacteria workup process involves the validation of the organism species and separating it into identifiable specie categories

Gram stain reaction, Cellular morphology or gram-positive or gram-negative bacteria

These are the bases for the categorization of organisms into cited broad categories

Catalase test

This is the test used to identify cellular morphology on gram-positive organisms

Oxidase test

This is the test used to identify cellular morphology on gram-negative organisms

MacConkey agar

This is the medium used for identified gram-negative rod or coccobacillus organisms

Catalase test, Oxidase test, MacConkey agar

These are helpful to the microbiologist in assigning the organism under observation to on of the primary cited categories

Identification of organisms based on common phenotypic traits shared with known members of the same genus or family

This is the clinical principle why microbiologist seem to be playing with the odds every day by finding the best biochemical fit and assigning the most probable organism identification

Neisseria species mixed testing results

This is the clinical rationale for the limitations of solely depending on flow charts for the identification process of organisms

HACEK organisms

This is the consideration of a microbiologist from an observed small-gram-negative rod in a Gram stain coming from a blood specimen with a patient having an endocarditis

Haemophilus spp., Aggregatibacter spp. Cardiobacterium spp., Eikenella corrodens, Kingella spp.

These are a group of gram-negative bacilli commonly known as the HACEK organisms

Differentiate microorganisms based on the ability to use acetamide as the sole source of carbon

This is the purpose of Acetamide utilization

Bacteria capable of growth on this medium produce the enzyme acylamidase

This is the principle of Actetamide utilization

5g of sodium chloride, 1g of ammonium dihydrogen phosphate, 1g of Dipotassium phosphate, 15g of agar, 0.8g of bromothymol blue indicator per 1000 mL, 10g of acetamide, 6.8 pH

This is the media contents of the agar or broth used in Actetamide utilization

Deaminates acetamide to release ammonia

This is the clinical significance of bacteria capable of growing on an Actetamide utilization medium to produce the enzyme acylamidase

Production of ammonia results in an alkaline pH

This is the clinical significance of bacteria producing acylamidase to deaminate acetamide to release ammonia

Causes the medium to change color from green to royal blue

This is the clinical significance of bacteria producing acylamidase to deaminate acetamide to release ammonia causing an alkaline power of hydrogen

Inoculate acetamide slant with a needle using growth from an 18- to 24-hour culture

This is the first methodical step for Acetamide utilization

Bacterial growth will be too heavy

This is the methodological significance of not inoculating from a broth culture instead of an 18-24 hour culture in Acetamide utilization

Incubate aerobically at 35-37 degrees Celsius for up to 4 days

This is the second methodical step for Acetamide utilization

Equivocal

This pertains to test results that could not be interpreted as Positive or Negative

Equivocal Acetamide slant

This is the methodological significance of reincubating an Acetamide slant for an additional 2 days

Reincubate for an additional 2 days

This is the number of days in reincubating an equivocal Acetamide slant

Blue color

This is the expected color result for a Positive Acetamide utilization

No color change

This is the expected color result for a Negative Acetamide utilization

Deamination of the acetamide

This is the methodological rationale for a blue colored result in Acetamide utilization

Bacterial growth without a color change may indicate a positive test result

This is the limitation of Acetamide utilization in bacterial growth results (?)

If further incubation results in no color change, repeat test with less inoculum

This is the limitation of Acetamide utilization in methodological incubation of inoculum (?)

Inoculum

This is the introduction of microbes into medium

Pseudomonas aeruginosa-growth, blue color

This is the organism and its expected result for the quality control of acetamide utilization to exhibit positive results

Escherichia coli-no growth, green color

This is the organism and its expected results for the quality control of acetamide utilization to exhibit negative results

Differentiate organisms based on ability to use acetate as the sole source of carbon

This is the purpose of Acetate utilization

Generally used to differentiate Shigella spp. and Escherichia coli

This is the general purpose of Acetate utilization

Organisms capable of using sodium acetate to grow on prepared medium

This is the clinical principle of Acetate utilization

Resulting in an alkaline pH

This is the clinical significance of organisms using sodium acetate for growth (?)

Turns the indicator from green to blue

This is the clinical significance of a Acetate utilization medium resulting to an alkaline pH

2g of Sodium acetate (NaC2H3O2), 0.1g of Magnesium sulfate (MgSO4), 1g of Ammonium dihydrogen phosphate, 20g of agar, 0.8g of bromothymol blue indicator per 1000 mL, pH 6.7

This is the media contents of the agar or broth used in Acetate utilization

Inoculate slant lightly with a straight inoculating needle from an 18- to 24-hour culture

This is the first methodological step of Acetate utilization

Bacterial growth will be too heavy

This is the methodological significance of not inoculating from a broth culture instead of an 18-24 hour culture in Acetate utilization

Incubate at 35-37 degrees Celsius for up to 7 days

This is the second methodological step of Acetate utilization

Medium becomes alkalinized, blue color

This is the expected positive result in Acetate utilization

No growth or growth with no indicator change to blue

This is the expected negative result in Acetate utilization

Some strains of E. coli may use acetate at a very slow rate or not at all

This is the limitation of Acetate utilization

Resulting in a false negative in the identification process

This is the clinical rationale why Acetate utilization could be limited because of E. coli

Escherichia coli-growth, blue color

This is the organism and its expected results for the quality control of Acetate utilization to exhibit positive results

Shigella sonnei-small amount of growth, green color

This is the organism and its expected results for the quality control of Acetate utilization to exhibit negative results

Test used in conjunction with Gram stain to distinguish aerobic gram-positive rods or coccobacilli

This is the test purpose of L-Alanine-7-amino-4-methylcourmarin (Gram-Sure)

Aerobic gram-positive rods or coccobacilli may appear gram-negative or gram-variable

This is the clinical rationale for distinguishing aerobic gram-positive rods or coccobacilli in L-Alanine-7-amino-4-methylcourmarin (Gram-Sure)

L-Alanine-7-amino-4-methylcourmarin

This is the compound impregnated in a commercially prepared disk, Remel-Thermo Fisher Scientific in Gram Sure

Remel-Thermo Fisher Scientific, Lenexa, KS

This is the commercially prepared disk impregnated with L-Alanine-7-amino-4-methylcourmarin and hydrolyzed by aminopeptidase

Gram-negative organisms produce an aminopeptidase

This is the principle used in L-Alanine-7-amino-4-methylcourmarin (Gram Sure)

Capable of hydrolyzing the reagent in the disk

This is the clinical rationale of aminopeptidase in L-Alanine-7-amino-4-methylcourmarin (Gram Sure)

Forming a blue fluorescent compound that is visible under long-wave UV light

This is the clinical rationale of hydrolyzing L-Alanine-7-amino-4-methylcourmarin with aminopeptidase in Remel-Thermo Fisher Scientific, commercial disk

Inoculate a pure colony of overnight growth to 0.25 mL of demineralized water in a clean 12 by 72 mm test tube

This is the first methodological step in L-Alanine-7-amino-4-methylcourmarin (Gram Sure)

16-18 hours after initial culture

This is the duration of the overnight growth of the organism in L-Alanine-7-amino-4-methylcourmarin (Gram Sure)

Place a Gram-Sure disk in the emulsion

This is the second methodological step in L-Alanine-7-amino-4-methylcourmarin (Gram Sure)

Incubate emulsifying Gram-Sure disk at room temperature for 5-10 minutes

This is the third methodological step in L-Alanine-7-amino-4-methylcourmarin (Gram Sure)

Observe blue fluorescence by placing the tube under long-wave UV light

This is the fourth methodological step in L-Alanine-7-amino-4-methylcourmarin (Gram Sure)

Fluorescent or blue

This is the expected result for aerobic, gram-negative rods and coccobacilli

Colorless

This is the expected result for aerobic, gram-positive rods and coccobacilli

Obligate anaerobic organisms may fail to give expected results

This is the limitation in L-Alanine-7-amino-4-methylcourmarin (Gram Sure)

Escherichia coli-blue fluorescence

This is the organism and its expected results for the quality control of L-Alanine-7-amino-4-methylcourmarin (Gram Sure) to exhibit positive results

Staphylococcus aureus-no fluorescence

This is the organism and its expected results for the quality control of L-Alanine-7-amino-4-methylcourmarin (Gram Sure) to exhibit negative results

Presumptive identification and differentiation of beta-hemolytic group A streptococci from other beta-hemolytic streptococci, Distinguish staphylococci species from micrococci

This is the purpose(2) of Bacitracin Susceptibility

The antibiotic bacitracin inhibits the synthesis of bacterial cell walls

This is the principle of Bacitracin Susceptibility

0.04 units of bacitracin is impregnated into TaxoA disk and placed on an agar plate

This is the methdological principle of the utility of bacitracin in Bacitracin Susceptibility

Allowing the antibiotic to diffuse into the medium and inhibit the growth of susceptible organisms

This is the methodological rationale of impregnating bacitracin into TaxoA disk and placed on an agar plate

Examined for zones of inhibition surrounding the disks

This is the clinical observation to be made after incubation on the inoculated plates of Bacitracin Susceptibility

Using an inoculating loop, streak two or three suspect colonies of a pure culture onto a blood agar plate

This is the first methodological step of Bacitracin Susceptibility

Using heated forceps, place a bacitracin disk in the first quadrant, gently tap the disk to ensure adequate contact with agar surface

This is the second methodological step of Bacitracin Susceptibility

First quadrant

This is the area of heaviest growth in the blood agar plate of Bacitracin Susceptibility

Look for a zone of inhibition around the disk

This is the fourth methodological step of Bacitracin Susceptibility

Incubate the plate for 18-24 hours at 35-37 degrees Celsius in ambient air for staphylococci and in 5-10% CO2

This is the third methodological step of Bacitracin Susceptibility

Streptococci differentiation

This is the methodological rationale of third methodological step of Bacitracin Susceptibility

Any zone of inhibition greater than 10 mm; susceptible

No zone of inhibition; resistant

Performance depends on the integrity of the disk

These are the limitations of Bacitracin Susceptibility

Proper storage and expiration dates should be maintained

This is the resolution for the limitation of Bacitracin Susceptibility

Streptococcus pyogenes-susceptible, Micrococcus luteus-susceptible

This is the organism and its expected results for the quality control of Bacitracin Susceptibility to exhibit positive results

Streptococcus agalactiae-resistant, Staphylococcus aureus-resistant

This is the organism and its expected results for the quality control of Bacitracin Susceptibility to exhibit negative results

Presumptive identification of enterococci and organisms in the Streptococcus bovis group; Differentiates enterococci and group D streptococci from non-group D viridands streptococci

This is the purpose(2) of Bile Esculin Test

Inhibition of Gram-positive bacteria, Growth of organisms capable to hydrolyze esculin to esculetin

This is the principle of Bile Esculin Test

4% bile

This is the percentage composition of bile used in Bile Esculin Test

Reacts with Fe3+ and forms a dark brown to black precipitate

This is the clinical significance of esculetin in Bile Esculin Test

11g of beef extract, 34.5g of enzymatic digest of gelatin, 1g of esculin, 2g of ox bile, 0.5g of ferric amonium citrate, 15g of agar per 1000 mL, pH 6.6

This is the medium composition of the agar slant used in Bile Esculin Test

Inoculate one to two colonies from an 18-24 hour culture onto the surface of the slant

This is the first methodological step in Bile Esculin Test

Incubate at 35-37 degrees Celsius in ambient air for 48 hours

This is the second methodological step in Bile Esculin Test

Growth and blackening of the agar slant

This is the expected positive result of Bile Esculin Test

Growth and no blackening of medium (not shown)

This is the expected negative result of Bile Esculin Test

Some organisms may grow poorly or not at all on this medium, because of nutritional requirements

This is the limitation of Bile Esculin Test

Enterococcus faecalis-growth, black precipitate

This is the organism and its expected results for the quality control of Bile Esculin Test to exhibit positive results

Escherichia coli-growth, no color change; Streptococcus progenes-no growth, no color change

This is the organism and its expected results for the quality control of Bile Esculin Test to exhibit negative results

Differentiates Streptococcus pneumoniae from alpha-hemolytic streptococci

This is the purpose of Bile Solubility Test

Bile or a solution of a bile salt rapidly lyses pneumococcal colonies

This is the principle of Bile Solubility test

Sodium deoxycholate

This is an example of a bile salt used in the bile solubility test

Amidase

This is the intracellular autolytic enzyme wherein the presence of it causes the intensity of lysis of organism in Bile Solubility Test

Lowers the surface tension between the bacterial cell membrane and medium

This is the clinical rationale of bile salts

Accelerates the organism's natural autolytic process

This is the clinical rationale of lowering the surface tension between the bacterial cell membrane and medium