Identifying an Unknown Staphs, Strep, or Enteric

Overview

This lab should give you the background information and techniques you will need to successfully perform biochemical tests in order to 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.

  • Each pair will receive one unknown organism to identify. You will conduct tests appropriate for your organism to determine genus and species identification.
  • Each pair may have to present information on the specific organism they identified including: test results, where that organism is part of the normal flora, when and where that organism becomes a pathogen, possible diseases the organism causes.
  • 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?

Work Flow

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Staphs

Staphylococcus species are normal flora widespread over the body surface. They are also important pathogens. Some of the most common diseases caused by Staphylococcus species include: impetigo, toxic shock syndrome, bacteremia, endocarditis, folliculitis furuncle (boils), and osteomyelitis (bone abscesses). Many species of Staphylococcus have the ability to form biofilms which can then colonize structures such as medical catheters, stents, heart valves, prostheses, shunts, and valves.

The clinically significant species are generally separated into coagulase-positive staphs (S. aureus) and coagulase-negative (CoNS) staphs (S. epidermidis, S. haemolyticus, and S. saprophyticus).

Streps

Many members of the Streptococcus genus are normal flora to the mouth, nose, and throat. The genus Streptococcus is a complex group causing a wide range of diseases such as: rheumatic fever, impetigo, pharyngitis, laryngitis, toxic shock syndrome, scarlet fever, and endocarditis.
Streptococci are often classified based on hemolysis which can be seen by their reaction on blood agar. Alpha hemolytic species produce alpha-hemolysin which reduces hemoglobin (red) to methemoglobin (green) causing a brownish or greenish zone around the colony. Beta hemolytic species produce a hemolysin that forms a clear zone around the colony, indicating complete lysis of red blood cells. Gamma hemolytic species are non-hemolytic, having no apparent effect on red blood cells.

Enterics

The Gram negative enterics are important both as natural flora in the intestinal tract and as pathogens of disease in the gastrointestinal tract and other sites. Four main families with numerous genera and species comprise the Gram negative enteric: Enterobacteriacea, Pseuodmonadaceae, Vibrionaceae, and Camplyobacteraceae. You will only be working with organisms from the first two families.

Lab Procedure

We have included the basic procedure for doing many common biochemical tests below. You will find more specific procedures for specific 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.

Biochemical Tests for Staphylococcus Organisms

Table 1: Brief Description of Biochemical Tests for Staphylococcus Organisms.

Test Brief Instructions Probable Results
TSA General Maintenance Media for Staphs Determine macromorphology
Gram Stain To confirm culture purity Staphs & Streps are Gram positive; Enterics are Gram negative
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.
Coagulase Add a loop-full or 0.5mL of a pure culture to 0.5mL rabbit plasma. Gently rotate tube to mix, do not shake. Incubate for 24 hours at 37°C. Presence of clot indicates S. aureus
Novobiocin Antibiotic Disk Sensitivity Dilute colonies from a pure culture into sterile saline to a 0.5 McFarland standard. Swab half the surface of a blood agar plate. Place a novobiocin disk lightly onto the surface. Incubate for 24 hrs at 37°C. A zone of growth inhibition ≤16 mm in diameter in a coagulase(-) staph is indicative of S. saprophyticus. See probable results table 2 below.
Hemolysis Streak the other half of the blood agar plate to check for hemolysis. Stab into the agar surface at the last part of your streak. Incubate 24 hrs in O2. Beta hemolysis is indicative of S. aureus. See probable results table 2 below.

Table 2: Probable Results for Staphylococcus Organisms

Hemolysis - Blood Agar

Intended Use

Blood agar is used to support the growth of fastidious organisms and to determine the type of hemolysis (destruction of red blood cell walls) an organism produces.

Principle

Blood agar is a rich medium that has been supplemented with fresh 5-10% blood. The hemolytic response can be dependent upon the type of blood. Sheep blood is commonly used, but some organisms require rabbit or bovine blood.

Test Procedure

  1. Streak a plate of blood agar for isolation.
    • Optional: Do your last streak with a needle and poke into the agar. This usually gives clear, reliable zones of beta hemolysis and is especially important to see the effects of streptolysin O which is oxygen labile. See page 84 of the Difco/BBL Manual.
  2. Incubate the plates at 37°C for 24-48 hours. Strep organisms should be incubated in the CO2 incubator.
    • The plate will be a brownish red color after 48hours.

Results

You can differentiate four types of hemolysis by the appearance of the agar.

  • Beta hemolysis is indicated by a clear colorless zone surrounding the colonies. There has been total lysis of the red blood cells.
  • Alpha hemolysis is indicated by a small zone of greenish to brownish discoloration of the media. This is caused by the reduction of hemoglobin to methemoglobin and its subsequent diffusion into the surrounding medium.
  • Alpha prime hemolysis is indicated by a zone of complete hemolysis, surrounded by a zone of partial hemolysis, a pink halo. This pattern can be easier to see if you scrape off the colony.
  • Gamma hemolysis is indicated by no change in the media.

Limitations

  • The patterns of hemolysis can vary with the incubation atmosphere and the type of blood in the media.
  • Some Staph organisms will only show hemolysis after they have been refrigerated following incubation.

Coagulase Test

Intended Use

Differentiates Staphylococcus aureus from other Staphylococcus species.

Principle

The coagulase test detects the presence of free and bound staphylcoagulase. This enzyme is excreted extracellularly by human strains of Staph. aureus. The mechanism of action is unknown.

Test Procedure

  1. Thaw a tube of 0.5 mL rabbit plasma.
  2. Inoculate a loop-full of organism into the tube. Chose a well isolated colony.
  3. Ideally you should incubate the tube at 35°C for 4 hours checking every 30 minutes for clot formation. We incubate them overnight and put them in the refrigerator until the next lab period with comparable results.
  4. Check for clot formation.
  5. Dispose of the tube in the biohazard container.

Results

The formation of a clot in the bottom of the tube is considered a positive result. The clot will not move as you tilt the tube. Unclotted plasma will flow in the tube.

Limitations

  • Methicillin resistant Staph. aureus have reduced clumping factor.
  • Do not shake or agitate the tube as this could break up the clot.
  • Some staphylococci strains produce fibrolysin after prolonged incubation at 35°C that can break up the clot resulting in false negative. Incubate the tube overnight at room temperature if you do not get a clot in 4 hours.
  • Some other rarely encountered staph species are also coagulase positive by the tube method.

Biochemical Tests for Streptococcus Organisms

Table 3: Brief Description of Biochemical Tests for Streptococcus Organisms.

Test Operating Instructions Probable Results
BH General Maintenance Media for Streps Determine macromorphology
Gram Stain To confirm culture purity Staphs & Streps are Gram positive; Enterics are Gram negative
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.
Optochin
Bacitracin
SXT		 Use your 0.5 McFarland standard to swab half the surface of a blood agar plate. Evenly place one of each disk on the swabbed agar surface. Any zone of inhibition around the Bacitracin disk is indicative of S. pyogenes. See probable results table 4 below.
Hemolysis Streak the other half of the plate to check for hemolysis. Stab into the agar surface at the last part of your streak. Incubate for 24 hrs in CO2. Beta hemolysis is indicative of S. pyogenes and S. agalactiae (sometimes). See probable results table 4 below.
Salt Tolerance Lightly inoculate broth. Loosely cap and incubate for 24-48 hours in CO2. Yellow color change indicative of Enterococcus faecalis. See probable results table 4 below.
Bile Esculin Streak the surface of the slant. Leave the cap loose. Incubate for 24-48 hours in CO2. Blackening of the agar is indicative of S. bovis and S. faecalis. See probable results table 4 below.

Table 4: Probable Results for Streptococcus Organisms

Bacitracin/SXT Sensitivity

Intended Use

Bacitracin differential disks are used to presumptively identify Group A, beta-hemolytic streptococci from other beta-hemolytic streptococci. The combination of SXT sensitivity increases the accuracy of the results.

Principle

Bacitracin is an antibiotic isolated from Bacillus subtilis. It inhibits cell wall synthesis mainly through inhibiting the biosynthesis of peptidoglycan. SXT inhibits folate metabolism which interferes with bacterial DNA synthesis. Group A, beta-hemolytic streptococci are more sensitive to bacitracin than other beta-hemolytic streptococci.

Test Procedure

The standard protocol has been modified for our lab.

  1. Using a loop, select 3-4 well isolated colonies, ideally from an 18-24 hour culture. Transfer to a small amount of sterile water.
    • Adjust the turbidity to 0.5 McFarland standard.
  2. Use the procedure outlined in antimicrobial susceptibility testing to swab the entire plate to obtain confluent growth.
  3. Visually divide the plate in thirds, place a bacitracin and SXT in their section of the plate. Using sterile forceps or a swab, lightly but firmly press the disks to the agar surface to adhere them.
    • Save the other section for the optochin disk.
  4. Invert the plates and incubate them for 18-24 hours at 35°C in 5-10% CO2.
  5. Incubate another 24 hours if the results are negative.

Results

  • Any zone of inhibition around the disk is considered sensitive (S).
  • No zone of inhibition with growth up to the disk is considered resistance (R).

This table is from MacFaddin, Biochemical Tests for Identification of Medical Bacteria.

Bacitracin SXT Presumptive ID
S R Group A b-streptococci
R R Group B b-streptococci
R S Not Group A or B b-streptococci
S S Rule out Group A or B with serologic tests

Limitations

  • Only beta-hemolytic streptococci should be tested.
  • While this test is accurate it is not highly specific. Other biochemical or serological tests are required for accurate identification.
  • The growth should be confluent. Too light of a growth could cause some non-group A streptococci to appear susceptible to bacitracin.

Hemolysis - Blood Agar

Intended Use

Blood agar is used to support the growth of fastidious organisms and to determine the type of hemolysis (destruction of red blood cell walls) an organism produces.

Principle

Blood agar is a rich medium that has been supplemented with fresh 5-10% blood. The hemolytic response can be dependent upon the type of blood. Sheep blood is commonly used, but some organisms require rabbit or bovine blood.

Test Procedure

  1. Streak a plate of blood agar for isolation.
    • Optional: Do your last streak with a needle and poke into the agar. This usually gives clear, reliable zones of beta hemolysis and is especially important to see the effects of streptolysin O which is oxygen labile. See page 84 of the Difco/BBL Manual.
  2. Incubate the plates at 37°C for 24-48 hours. Strep organisms should be incubated in the CO2 incubator.
    • The plate will be a brownish red color after 48hours.

Results

You can differentiate four types of hemolysis by the appearance of the agar.

  • Beta hemolysis is indicated by a clear colorless zone surrounding the colonies. There has been total lysis of the red blood cells.
  • Alpha hemolysis is indicated by a small zone of greenish to brownish discoloration of the media. This is caused by the reduction of hemoglobin to methemoglobin and its subsequent diffusion into the surrounding medium.
  • Alpha prime hemolysis is indicated by a zone of complete hemolysis, surrounded by a zone of partial hemolysis, a pink halo. This pattern can be easier to see if you scrape off the colony.
  • Gamma hemolysis is indicated by no change in the media.

Limitations

  • The patterns of hemolysis can vary with the incubation atmosphere and the type of blood in the media.
  • Some Staph organisms will only show hemolysis after they have been refrigerated following incubation.

Salt Tolerance Broth

Intended Use

Salt tolerance broth is intended to differentiate non-beta-hemolytic strains of streptococci.

Principle of Use

Brain Heart Infusion (BHI) broth is supplemented with 6.5% sodium chloride and bromcresol purple as a pH indicator. The indicator is included to make reading the test results easier. The broth also includes dextrose. The fermentation of dextrose (glucose) results in the production of acid. This changes the pH of the media causing the media to turn from purple to yellow.

Test Procedure

  1. Select no more than 2-3 colonies (preferably from an overnight culture) to inoculate a tube of salt tolerance broth.
    • It is important to lightly inoculate the tube otherwise you may get a false positive.
  2. Loosen the cap and incubate aerobically for 24 hours at 37°C.
  3. Continue incubation up to 72 hours if you get a negative result at 24 hours.

Results

A positive reaction is indicated by obvious turbidity in the media with or without a color change. A negative result is indicated by no growth after 72 hours. Enterococcus spp. typically changes the media color within 24 hours.

Limitations

  • Many staphylococci can grow in media containing 10% salt. Mannitol salt agar has 7.5% salt.
  • Salt tolerance media was intended to differentiate catalase negative gram-positive cocci. Be sure to perform a catalase test before you proceed with the salt tolerance broth test.
  • Other species of catalase negative gram-positive organisms can grow in this media.

Biochemical Tests for Enteric Organisms

Table 5: Brief Description of Biochemical Tests for Enteric Organisms.

Test Brief Instructions Probable Results
TSA General Maintenance Media for Staphs Determine macromorphology
Gram Stain To confirm culture purity Staphs are Gram positive
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.
Mac Streak for isolation. Incubate 24-48 hrs at 37°C. See probable results table below.
EMB Streak for isolation. Incubate 24-48 hrs at 37°C. See probable results table below.
Citrate Streak surface only. Incubate loosely-capped 24-48hrs at 37°C. See probable results table below.
TSI With a needle pick the center of a well isolated colony. Stab the center of the tube to within 3-5 mm of the bottom. Withdraw the needle and lightly streak the surface of the slant. Incubate for 24 hrs at 37°C. See probable results table below.
Urea Heavily inoculate a tube of urea broth. Shake tube to distribute organisms. Incubate for 24-48 hrs at 37°C.  
Simmons Citrate Agar Image

Simmons Citrate Agar Slant

Simmons Citrate Agar Slant

Principle

Used for the differentiation and identification of Enterobacteriaceae on the basis of citrate utilization, citrate being the sole carbon source.

Purpose

Colonies capable of utilizing citrate as a carbon source produce a local increase in pH, changing the color of the medium from green to blue. Only citrate positive organisms will grow on this medium.

Test Procedure

  1. Inoculate the organism directly onto the surface of a Citrate slant.
  2. Incubate aerobically at 35-37°C.
  3. Examine for growth and color change after 18-24 hours of incubation.

Interpretations

Good growth with the medium color turning blue indicative of Enterobacter aerogenes and Salmonella choleraesuis.

Eosin Methylene Blue Agar Image

Eosin Methylene Blue (EMB) Agar

Eosin Methylene Blue (EMB) Agar

Principle

A differential plating medium for the detection & isolation of the gram-negative enteric bacteria.

Purpose

  • To aid in the differentiation of lactose fermenting bacteria from non-lactose fermenting bacteria.
  • To aid in the differentiation of Enterobacter aerogenes and Escherichia coli.

Test Procedure

  1. Inoculate the organism directly onto the surface of an EMB agar plate and streak for isolation.
  2. Incubate inoculated plate aerobically at 37°C.
  3. Examine for growth after 18-24 hours of incubation.

Interpretations

  • Coliforms that utilize the lactose and/or sucrose are blue/black with a greenish metallic sheen. Indicative of Escherichia coli.
  • Coliforms such as Klebsiella pneumoniae have mucoid colonies that may be purple and/or exhibit a green metallic sheen.
  • Good to excellent, colorless colonies indicative of Proteus vulgaris, Salmonella choleraesuiss, and Shigella spp.
MacConkey Agar Image

MacConkey (Mac) Agar

MacConkey (Mac) Agar

Principle

  • A differential plating medium recommended for use in the isolation and differentiation of lactose-fermenting organisms from lactose non-fermenting gram negative enteric bacteria.
  • Selectivity of the medium is due to the presence of crystal violet and bile salts which markedly to completely inhibit the growth of gram positive organisms.

Purpose

  • Organisms capable of fermenting lactose produce a localized pH drop which, followed by the absorption of neutral red, imparts a red/pink/purple color to the colony. A zone of precipitated bile may also be present due to this localized drop in pH.
  • Organisms that do not ferment lactose remain colorless and translucent. They are easily detected by transmitted light and appear as colorless colonies against a red background.

Test Procedure

  1. Inoculate the organism directly onto the surface of a MacConkey agar plate and streak for isolation.
  2. Incubate inoculated plate aerobically at 35-37°C.
  3. Examine for growth after 18-24 hours of incubation.

Interpretation

  • Good to excellent growth, red/pink/purple colonies with bile precipitate indicative of Escherichia coli.
  • Good to excellent growth, red/pink/purple colonies without bile precipitate indicative of Enterobacter aerogenes.
  • Good to excellent, colorless colonies without bile precipitate indicative of Proteus vulgaris, Salmonella typhimurium, and Shigella spp.