General Biochemical Tests

Identifying Unknown Bacteria

Overview

This lab should help give you the background information and techniques you will need to successfully perform general biochemical tests in order to help identify unknown bacterial samples. The micro lab website, your textbook, the web and assorted books available in lab will be the reference materials necessary for you to successfully complete the next several weeks of lab work.

  • You will perform general biochemical tests on an unknown organism.
  • For each biochemical test you perform, make sure to record the following in your lab book:
    • What does a positive test result look like?
    • What is the biochemical basis of the test?

Lab Procedure

We have included the basic procedure for doing each biochemical test in the table below. You will find more specific procedures for each biochemical test on the following pages. More complete information on selective & differential media can be obtained by consulting the Difco manuals in lab. You will need to look up the individual test for a more detailed description, including the biochemical basis of each test.

Table 1: Brief Description of general tests and probable results.

Test Brief Instructions Probable Results
TSA/BHI Staphs/Enterics on TSA; Streps on BHI Determine macromorphology
Gram Stain To confirm culture purity Staphs/Streps (Gram+), Enterics (Gram-)
Motility Stab with a needle straight in and straight out of the center of the tube half way down.
Incubate for 24 hours at 37°C.
Staphs/Enterics in O2; Streps in CO2.		 Motile organisms have obvious growth away from inoculation area; Non-motile organisms grow only in inoculation area.
McFarland Standard Dilute your organism in a tube of sterile water to obtain a turbidity equivalent to a 0.5 McFarland test standard. Hold your diluted tube and the 0.5 McFarland test standard against the black-lined McFarland reference card to accurately rate the turbidity.
FTM Use a sterile transfer pipette to add 1 mL of your McFarland standard organism in the middle of the tube. Cap tightly; do not jostle. Incubate for 24 hours at 37°C. Strict aerobes will grow near the top of the media; Facultative anaerobes will grow throughout; Strict anaerobes will grow near the bottom.
Catalase Transfer a well-isolated colony to a clean slide & add 1 drop of 3% H2O2. Do not reverse the order & do not mix. Observe for immediate bubble formation. Staphs/Enterics are catalase positive; bubble formation should occur. Note: do not take colony from a blood plate.
Oxidase Add a few drops of oxidase reagent onto Whatman filter paper. Smear with a loop-full of organisms. Use colonies from low glucose, non-selective media.  Look for appearance of purple color within 30 seconds. 

Motility Test Medium

Intended Use

  • Used for detecting motility of microorganisms.

Principle

Motility is apparent by the presence of diffuse growth away from the line of inoculation.

Test Procedure

  1. Inoculate with growth from an 18-24 hour culture by stab inoculation with a needle.
  2. Incubate at a temperature and duration appropriate for the organism being tested.
  3. Examine tubes for growth and signs of motility.

Results

  • Motility is apparent by the presence of diffuse growth away from the line of inoculation.
  • Non-motile organisms only grow along the line of inoculation.

Limitations

  • The motility of Proteus spp. is temperature dependent.
  • Due to the temperature dependency of motility in some organisms, a negative tube should be incubated an additional 5 days at a lower temperature of 22-25°C.

Fluid Thioglycolate Medium (FTM)

Tests the oxygen requirements of different microorganisms.

Intended Use

Characterizes microbes according to their oxygen requirements

  • Obligate aerobes
  • Facultative anaerobes
  • Obligate anaerobes

Principle

Various types of bacteria require various oxygen (or oxygen-free) environments to grow in.

Test Procedure

  1. Dilute your organism in a tube of sterile water to obtain a turbidity equivalent to the 0.5 McFarland test standard. Hold your diluted tube and the 0.5 McFarland test standard against the black-lined McFarland reference card to accurately rate the turbidity.
  2. Using a sterile 1mL pipette, place 1 mL of organism into the middle of the tube.
  3. Cap tightly; do not jostle.
  4. Incubate for 24 hours at 37°C.

Results

  • Strict (obligate) aerobes grow at the surface of the medium where there is a high concentration of oxygen.
  • Obligate anaerobes grow near the bottom of the broth tube where there is no oxygen.
  • Facultative anaerobes grow best where more oxygen is present, but growth will occur throughout the broth tube.

Catalase

Intended Use

  • Differentiates Streptococcus (-) from Micrococcus (+)
  • Differentiates Staphylococcus (V+) and Bacillus (+) from Clostridium (-)

Principle

Hydrogen 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

  1. Transfer a well isolated colony to a clean glass slide and add 1 drop of 3% H2O2.
    •  Do not reverse the order and do not mix.
  2. Observe for immediate bubble formation.
  3. Use 15% H2O2 for the detection of catalase in anaerobes.

Results

  • The formation of bubbles is considered a positive result.

Limitations

  • Do not take your colony from a blood agar plate. The catalase present in the erythrocytes will give a false positive result.
  • H2O2 is unstable. You can do a quality control test of the H2O2 reagent by placing a drop on a blood agar plate. Vigorous bubbling should result.

Oxidase Biochemical Assay

Tests for the presence of the enzyme indophenol oxidase.

Intended Use

The 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.

Principle

Indophenol oxidase, in the presence of atmospheric oxygen, oxidizes the phenylenediamine oxidase reagent to form a dark purple compound, indophenol.

Test Procedure

  1. Have your instructor or IA crush the ampule inside the dropper.
  2. Tap bottom on tabletop a few times. Then invert for convenient drop-by-drop dispensing of reagent
  3. Preparation for testing:
    • Colonies to be tested must be isolated from other colonies
    • The use of fresh isolates (18-24 hr cultures) is recommended for routine testing.
    • If refrigerated, cultures must be allowed to reach room temperature prior to testing
  4. Performing the test – Filter Paper Method
    • Add a few drops of oxidase test reagent to a strip of filter paper (Whatman No. 1 or equivalent).
    • Streak a loopful of bacteria onto the reagent-saturated paper with a platinum loop or wooden applicator stick. Use of steel of nichrome loops may cause false-positive reactions

Results

Positive 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

  • Allow up to 30 seconds for a positive reaction.
  • Any delayed reactions should be considered negative.
  • Do not add excess reagent, at it may cause the reaction to fade on oxides-positive organisms.
  • Steel loop, nichrome loop, and wire loop containing iron may give a false-positive reaction. A platinum loop or wooden applicator stick is recommended.