It often becomes necessary to identify unknown bacteria. In this exercise you will use the techniques used in the previous lab exercises to characterize and identify an unknown species of bacteria. The species may or may not be one you have studied previously. This exercise will be graded as described in the "RESULTS" section.
Glucose fermentation is shown by a yellow color with or without gas in the glucose broth with the Durham tube and in the TSI tube. GluA(+) indicates glucose fermentation with acid, GluG(+) indicates glucose fermentation with gas.
Lactose fermentation is shown by the pink or red colonies in the MacConkey agar plate and in the TSI tubes. LacA(+) indicates lactose fermentation with acid, LacG(+) indicates lactose fermentation with gas. Lactose fermentation can also be shown by using a lactose broth similar to the glucose broth above. The MacConkey agar plate also inhibits growth of Gram positive bacteria.
The TSI reactions include:
No growth or no fermentation (TSI-) - red slant and red butt. Growth in TSI, TSI(+).
Glucose fermentation - red slant, yellow butt (B+) due to acid production.
Lactose and/or sucrose fermentation - yellow slant (S+) and yellow butt (B+), both due to acid production.
Gas produced (G+) - split in the agar.
Hydrogen sulfide produced (H2S+) - black in butt.
If the enzyme gelatinase is produced, gelatin will be hydrolyzed (Gel+), shown when the gelatin remains liquid when cooled.
Most bacteria produce the enzyme catalase (Cat+) which breaks down hydrogen peroxide (H2O2) to protect from the harmful effects of oxygen. The breakdown results in foaming when H2O2 is added to a colony of H2O2(+) bacteria.
Bacteria which use citrate as a source of carbon turn citrate agar blue, citrate(+).
Bacteria which carry out respiration usually contain cytochrome C. The oxidase test indicates the presence of cytochrome C (Ox+)
Indole(+) bacteria produce indole from tryptophan.
Bacteria are subdivided into four Divisions or Phyla based on wall characteristics. Each Division is subdivided into Sections which contain Families, Genera, and Species. The following is a modified classification of some of the more common bacteria encountered in these lab exercises.
DIVISION - ARCHAEOBACTERIA
Cell walls present but they lack peptidoglycan; not studied in this laboratory exercise.
DIVISION - MYCOPLASMAS
Cell walls absent; not studied in this laboratory exercise.
DIVISION - GRAM NEGATIVE BACTERIA
SECTION - SPIROCHETES
SECTION SPIRAL AND CURVED BACTERIA
SPIRAL FAMILY (SPIRILLACEAE) - curved rods, some with many turns; motile; aerobic, facultatively anaerobic, or anaerobic; cannot ferment carbohydrates; ox(+); indole(-).
Genus Aquaspirillum (formerly Spirillum) - obligate aerobe or microaerophilic; Gel(-); H2S(+); cat(+).
A. itersonii - growth in TSI and MacConkey agar.
A. serpens - Gel, TSI, and MacConkey agar variable.
A. sinuosum - no growth in TSI and MacConkey agar.
SECTION AEROBIC RODS - no fermentation, therefore GluA(-), GluG(-) and LacA(-) and LacG(-); respiration only.
PSEUDOMONAD FAMILY (PSEUDOMONADACEAE) - cat(+); usually ox(+); Gel variable.
Genus Pseudomonas - motile; Ox variable; TSI: S(-) B(-) G(-) H2S(-), often turns agar green.
P. aeruginosa - Ox(+); Gel(+).
Genus - Alcaligenes - motile; Gel(-); Ox(+).
A. fecalis - cells rods or cocci, usually single; motile; citrate(+).
SECTION - AEROBIC COCCI
NEISSERIA FAMILY (NEISSERIACEAE) - cells usually large obvious cocci; cells usually in pairs, some clumps; non-motile; most are Cat(+); most are Ox(+).
Genus Neisseria - cocci; aerobic or facultatively anaerobic; Cat(+); Ox(+).
N. sicca - GluA(+), H2S(+).
SECTION - FACULTATIVELY ANAEROBIC RODS
ENTERIC FAMILY (ENTEROBACTERIACEAE) - aerobic and facultatively anaerobic; fermentation, motility variable; GluA(+); Cat(+); Ox(-).
In general pathogenic enterics cannot ferment lactose.
Genus Escherichia - motile; lactose variable; TSI: S(+) B(+) G(+) H2S(-); Gel(-); Citrate (-); Indole(+), few other bacteria are Indole(+).
E. coli -
Genus Enterobacter - motile; ferments lactose; GluG(+) at 37 degrees C; Citrate(+).
E. aerogenes -
Genus Serratia - usually produce red colonies; motile; does not ferment lactose; Gel(+); Citrate(+).
S. marcescens -
Genus - Proteus - motile; does not ferment lactose; a small amount of gas from glucose fermentation; H2S variable; Gel variable; TSI variable; usually Indole (+).
P. vulgaris - GluG(+); Gel(+); H2S(+); Indole(+).
P. mirabilis - GluG(+); Gel(+); H2S(+); Indole(-).
Other members of the enterics include *Salmonella, Shigella; Klebsiella; Yersina.
DIVISION - GRAM POSITIVE BACTERIA
SECTION - COCCI
MICROCOCCUS FAMILY (MICROCOCCACEAE) - cells usually grow in clumps; motility variable; aerobic or facultatively anaerobic; if glucose fermented, GluA(+); Cat(+).
Genus Micrococcus - often non-motile; obligate aerobe; GluA(-), GluG(-); Indole(-).
M. luteus - yellow colonies; Ox variable.
M. roseus - red colonies.
Genus Staphylococcus - non-motile; GluA(+); Gel(+).
S. aureus -
STREPTOCOCCUS FAMILY (STREPTOCOCCACEAE) - cells grow in chains or pairs; non-motile; fermentation; Cat variable.
Genus Streptococcus - Cat(-); GluA(+), GluG(-); usually Gel(-).
S. lactis - cells usually in pairs or short chains; grows at 10 degrees C, does not grow at 45 degrees C.
S. faecalis - cells usually in pairs or short chains; grows at 10 degrees C and 45 degrees C.
S. salvarius - cells usually in short to long chains; does not grow at 10 degrees C, grows as 45 degrees C.
SECTION - ENDOSPORE‑FORMING RODS
BACILLUS FAMILY (BACILLACEAE) - motility variable; aerobic, facultatively anaerobic, anaerobic; Cat variable.
Genus Bacillus - motile; cells may grow in chains; facultatilve anaerobe; GluA(+), GluG(-); Cat(+).
B. subtilis - cells usually not in chains.
B. cereus - cells usually in chains.
Genus Clostridium - obligate anaerobes.
SECTION - NONSPORE PRODUCING RODS
LACTOBACILLUS FAMILY (LACTOBACILLACEAE) - cells usually single or chains; facultative anaerobic or anaerobic; non-motile; cat(-).
Genus Lactobicillus - fermentative metabolism but grows in air; GluA(+), GluG variable.
L. acidophilus - GluG(-).
CORYNEBACTERIUM FAMILY (CORYNEFORM GROUP) - irregular shaped rods.
Genus Corynebacterium - aerobic or facultatively anaerobic; Cat(+).
C. pseudodiphtheriticum - GluA(-).
C. xerosis - GluA(+).
SECTION - MYCOBACTERIA
MYCOBACTERIUM FAMILY (MYCOBACTERIACEAE) - slightly curved or straight rods; acid-fast; aerobic; non-motile.
M. smegmatis - no growth on MacConkey agar.
Using an unknown, determine:
A. Cultural characteristics by inoculating an agar plate, an agar slant, and a tube of nutrient broth.
B. Morphological characteristics by preparing a Gram stain.
C. Physiological characteristics including gelatin hydrolysis, fermentation of glucose, reactions in TSI and MacConkey agar, motility, and catalase, oxidase and indole reactions.
A. STOCK CULTURES
Unknown bacteria ‑ broth
1. Nutrient agar plates ‑ 1/student
3. Nutrient agar slants ‑ 3/student
4. TSI agar slants ‑ 1/student
5. Nutrient gelatin tubes ‑ 1/student
6. Nutrient broth tubes ‑ 1/student
7. Glucose broth Durham tubes with phenol red ‑ 1/student
8. Lactose broth Durham tubes with phenol red – 1/student
9. MacConkey agar plates ‑ 1/student
10. Citrate agar tubes ‑ 1/student
11. Motility medium tubes ‑ 1/student
12. Thioglycolate tube – 1/student
C. STAINS AND SOLUTIONS
1. Crystal violet
2. Gram's iodine
5. Tap water in wash bottles
6. Hydrogen peroxide solution
7. Oxidase reagent droppers
8. Indole reagent droppers
1. Inoculating loops and needles
2. Bunsen burners
3. Staining pans with racks ‑ 4/table
4. Microscope Slides
5. Ice in buckets
6. Filter paper strips
7. Sharpie Pens
You will be given a plate culture of one unknown species of bacteria. PUT THE NUMBER OF YOUR UNKNOWN IN THE BLANK ON YOUR ANSWER SHEET. Keep your unknown with your cultures. Do not return it to the unknown shelf. Complete the following. You are not expected to complete this entire exercise in this lab period.
This procedure requires:
(1) Nutrient agar plate
(2) Three nutrient agar slants. One may serve as a reserve stock to make a new working stock if the original unknown culture becomes contaminated.
(3) TSI agar slant
(4) Nutrient gelatin tube
(5) Nutrient broth tube
(6) Glucose broth in Durham tube with phenol red
(7) MacConkey agar plate
(8) Citrate agar tube
(9) Motility medium tube
(10) Thioglycolate tube
(11) Lactose broth in Durham tube with phenol red
1. Inoculate the nutrient agar plate using the streak plate isolation method.
2. Inoculate three nutrient agar slants, the MacConkey agar plate, and the citrate agar tube by streaking the surface of the agar with the inoculating loop.
3. Inoculate the TSI agar slant by stabbing the agar with the inoculating needle and streaking the surface of the slant as the needle is withdrawn.
4. Inoculate the nutrient gelatin tube, thioglycolate tube,and motility medium tube by stabbing with the inoculating needle.
5. Inoculate the nutrient broth tube, lactose broth tube and the glucose broth tube by inserting the loop into the broth.
B. COLONY CHARACTERISTICS
1. Next lab, examine isolated colonies on the nutrient agar plate, a nutrient agar slant, and the nutrient broth tube and determine:
The colony form, elevation, and margin on the nutrient agar plate.
The colony form on the nutrient agar slant.
The type of growth in the nutrient broth tube.
2. Draw a picture of your plate and describe your colony characteristics in "RESULTS".
This procedure requires:
(1) 1 or 2 clean microscope slides
(2) Crystal violet stain
(3) Gram's iodine stain
(4) Acetone alcohol
(5) Safranine stain
(6) Tap water in wash bottles for rinsing slides
(7) Unknown culture
Prepare a Grams stain of your unknown. As a backup, you may also prepare a simple stain of your unknown. If your results are unsatisfactory, you may repeat the staining using a culture from your slant or broth or streak plate prepared above.
1. Clean a blank microscope slides thoroughly. New slides from packages are probably already cleaned.
2. Label the slide.
3. Prepare a smear by transferring a loopful of your unknown broth culture onto the slide. If transferring from agar, place a loopful of water on the slide then stir a loopful of culture into the water.
4. Allow the slide to air dry.
5. Pass the slide through the flame several times to fix the smear.
6. Flood the smear with crystal violet for one minute.
7. Rinse each slide with tap water from the wash bottle.
8. Flood the smear with Grams iodine for one minute.
9. Rinse the slide with tap water.
10. Decolorize the smear by adding a few drops of acetone- alcohol for 10‑15 seconds.
11. Rinse the slide with tap water.
12. Flood the slide with safranin for one minute.
13. Rinse the slide with tap water.
14. Blot the slide dry between pages of the bibulous paper booklet.
15. Observe the slides under 10X, 40X, and 100X (oil immersion) magnification.
16. Determine cell shape, cell arrangement, and Gram stain. Recall that Gram positive cells are purple and Gram negative cells are red.
17. Draw a picture and describe the characteristics of your bacteria in "RESULTS".
D. PHYSIOLOGICAL CHARACTERISTICS
Next lab, examine the remaining media.
1. Place the tube of gelatin in ice for several minutes. If the gelatin remains liquid, hydrolysis has occurred.
2. Observe the tube of glucose lactose broth. If acid has been produced the tubes will appear yellow. If gas has been produced, a bubble will appear in the Durham tube.
3. Observe the tube of TSI agar. A yellow color indicates acid has been produced and a split in the agar shows the presence of gas. A black color indicates amino acids have been broken down. Acid production from glucose fermentation results in a red slant and yellow butt. Acid production from lactose or sucrose fermentation results in a yellow slant and butt.
4. Look for isolated colonies on the plate of MacConkey agar. Colonies which appear pink or red indicate lactose fermentation. Presence of bacteria indicates the bacteria are probably Gram negative.
5. Observe the tube of motility medium. If the bacteria are motile, red color will look fuzzy and spread away from the stab mark. If the bacteria are not motile, the red color will not look fuzzy and will be located right at the stab mark.
6. Add several drops of hydrogen peroxide solution to one of the agar slants and observe for foaming, which indicates a positive reaction.
7. Observe the color of the tube of citrate agar. If the bacteria use citrate the tube will appear blue and the reaction is positive. If the bacteria do not use citrate the tube will remain green.
8. Complete the oxidase test using bacteria from one of your agar slants as follows:
a. If the oxidase reagent dropper has been used by someone else continue with step c.
b. If the oxidase reagent dropper has not been used previously, hold the reagent dropper upright and point the tip away from you. The dropper contains an ampule. Squeeze the dropper in the middle with your thumb and forefinger to crush the ampule. Squeeze only once; further squeezing may cause glass from the ampule to puncture the plastic reagent dropper. Tap the bottom of the reagent dropper on the table top a few times.
c. Add a few drops of the oxidase test reagent to the slant and observe for a color changes within 10 to 30 seconds. A positive reaction will first turn pink then become dark purple.
9. Complete the indole test using bacteria from your remaining agar slant as follows:
a. Use the indole reagent dropper as described above.
b. Add a few drops of the indole test reagent to another slant and observe for a color changes within 10 to 30 seconds. A positive reaction will be a red color.
11. Observe the results of the thioglycolate tube. Growth only in the upper region and around the stab mark indicates aerobic bacteria. Growth only in the colorless part of the tube indicates anaerobic bacteria. Growth throughout the tube indicates facultatively anaerobic bacteria.
10. Complete table in "RESULTS". Write the number and species name of your bacterium in