PHOTOGRAPHIC ATLAS

BIOL203: Elementary Microbiology

By: Rose Alexander

April 25, 2018

 Lab 1: The Ubiquity and Culturing of Microorganisms
Introduction: Microorganisms are found everywhere on Earth in diverse environments. They can vary in
almost every aspect of their being including shape, size, cell structure, behavior, and metabolism. In this lab we
learned the basic techniques used by microbiologists to grow and describe microorganisms.
Exercise 1A: Inoculating Agar Plates
Purpose: To learn how to prepare agar plates and culture microorganisms from an environmental source.
Method:
1. Pour 10-20mL of melted TSA into sterile Petri dish (enough to cover the bottom)
2. Allow to cool for 10-15 minutes (solidified when TSA turns cloudy)
3. Wet a sterile cotton swab and collect a sample by wiping the wet swab over the surface
4. Wipe the swab over the TSA, return lid, and incubate
Results:

Figure 1: My environmental sample was obtained from the toilet seat of Stall 1 in the women’s bathroom of the Science I Building on the SUNY
Oneonta campus. This photo is evidence that the collected microorganism(s) do not grow when incubated at 20°C and that 37°C is their preferred
temperature or the temperature that is best for their growth.

Exercise 1B: Inoculating TSA Slants Aseptically

Purpose: To learn and use aseptic technique to prevent contamination when culturing microorganisms.
Method:
1. Wipe bench top with 70% alcohol from spray bottle
2. Gather materials and light Bunsen burner (remain within 12in of the flame while working)
3. Sterilize the inoculating loop in the fire until it is red hot (do NOT set down on the bench)
4. With free hand grab the stock culture and remove the top with the pinky finger of the hand holding the
inoculating loop (do NOT set the lid down)
5. Pass the opening of the stock culture tube through the flame
6. Insert inoculating loop into tube and touch to the glass to cool it (do NOT touch the TSA yet)
7. Touch the cooled loop to the culture
8. Remove loop, flame the opening of the stock culture, and replace lid
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 9. Pick up fresh TSA slant with free hand, remove lid with pinky, flame the opening, and inoculate the
TSA
a. For slants rub the loop on the surface, for broths just dip the loop in
10. Remove the loop, flame the opening of the new culture, and replace the cap
a. Flame the loop once more before setting it down on the bench
Results:

Figure 2: Three TSA slants were inoculated with different bacteria and the colored areas (red and yellow) are bacterial growth resulting from proper
inoculation.

Figure 3: From left to right - Serratia marcescens, Micrococcus luteus, Mixed culture of S. marcescens and M. luteus.

Exercise 1C: The Four Way Streak Method

Purpose: To learn how to do the four-way streak method on agar plates.

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Method:
1.
2. Wipe bench with 70% alcohol
3. Divide an agar plate into 4 quadrants
4. Use aseptic technique to pick up a sample of stock
culture
5. Smear the stock culture over the surface of quadrant 1
6. Flame the loop and cool against the glass
7. Run the loop in a line through quadrant 1 then smear the
sample over the surface of quadrant 2
8. Repeat steps 5-6 for quadrants 3 and 4
Results:

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 Lab 2: Cultural and Cellular Morphology
Introduction: Morphology, the shape, size, arrangement, structures, and characteristics of their colonies, is
used to identify species of microorganisms. Morphology can be observed by looking at the colonies or by
looking at the microorganisms using a microscope.
Exercise 2A: How to Describe Broth, Agar Slant, and Agar Plate Morphology Like a Microbiologist
Purpose: To use microbiology terminology to describe broth culture, agar slant colony, and single colony
morphology using the correct terms.
Method:
1. Observe the cultures and use the terms to describe them
Important Terms:

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Exercise 2B: Looking at Bacteria Under the Microscope with Simple Staining
Purpose: To learn how to prepare smears that will be stained and observed to describe the shape and
arrangement of the cells.
Method:
1. Making the smear
a. Place a drop of water on the bench and take a loopful to place on the slide
b. Collect a SMALL sample of the stock culture on the inoculating loop using aseptic technique
c. Work the sample into the water (the smear should be thin)
d. Allow the smear to air dry (a good smear will be slightly cloudy and see through)
e. Pass the slide through the Bunsen burner flame to heat fix the smear
2. Staining the smear
a. Add a couple drops of methylene blue to the slide
b. Let sit for 1 minute
c. Rinse the excess dye with distilled water
d. Blot the smear dry with Bilbous paper

3. Looking under the microscope

a. Center the smear on the stage and use the coarse then fine focus knob to bring the image into
focus
b. Switch to the 10X objective and adjust the focus using the fine focus knob
c. Repeat with the 40X objective
*Very little adjustment is needed with the microscope. If the image is lost, start from the
beginning to find it again
*Adjusting the light with the rheostat can be helpful to better visualize the slide

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Results:

Bacillus subtilis Micrococcus luteus

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 Lab 3: The Gram and Acid-Fast Stains
Introduction: Staining can be used for more than just visualizing microorganisms under the microscope. It can
actually be used to identify them. Differential staining uses more than one stain to differentiate between cells
based on their characteristics.
Exercise 3A: Performing the Gram Stain
Purpose: To learn how to perform the gram stain to differentiate between gram positive and gram-negative
species and observe their morphology.
Method:
1. Prepare and heat fix a slide of organisms
2. Place a couple drops of crystal violet stain on the smear and let sit for one minute
3. Wash off excess crystal violet with distilled water
4. Add a few drops of Gram's iodine and let sit for one minute
5. Wash off excess Gram's iodine with distilled water
6. Decolorize the cells by rinsing with 95% alcohol until the alcohol runs clear
7. Wash off excess alcohol with distilled water
8. Counterstain by adding a few drops of safranin and let sit for one minute
9. Wash off excess safranin with distilled water
10. Blot the slide dry with Bilbous paper and view under the microscope

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Results:

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 Lab 4: Bacterial Growth
Introduction: Each species of microorganism has a generation time. This is the time it takes for one cell to
undergo binary fission. The number of cells in each generation can be calculated using the equation 2 n.
Bacterial cells grow exponentially and go through four phases.

Exercise 4A: Estimating the Number of Organisms Using the Plate Count Technique
Purpose: To learn how to get a count of bacteria using the plate count technique.
Method:
1. Place 9mL of sterile water into a set of clean tubes
2. Add 1mL of stock culture into the first tube and pipette up and down to mix well. This represents the
1:10 or 10-1 dilution
3. Next take 1mL of the dilution you just made and add it to another tube. This tube is the 10 -2 dilution
4. Repeat step 3 until a 10 -7 dilution is made
5. Prepare spread plates of the 10-6 and 10-7 dilutions
a. Take 3 agar plates for each dilution
b. Aseptically dispense 1mL of the dilution on each plate
c. Dip a glass hockey stick into the dish of alcohol and burn off the alcohol in the flame
d. Cool the stick and use it to spread the dilution over the plate. Flame the stick in between uses
e. Put the lid on the plate and allow to sit for 10 minutes
f. Incubate the plates at 37°C
Results:
1 2 3 Average
10-6 14 11 3 9.3

Enumeration: 93,000,000 cfu/mL

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Exercise 4B: Gas Requirements for Microbial Growth
Purpose: To learn how to differentiate microorganisms based on their gas needs and how to grow anaerobic
microorganisms.
Method:
1. Take 2 TSA plates and divide them into 5 pie slices
2. Aseptically streak a sample of each culture over the surface of one of the pie slices
3. Place one plate in the anaerobic jar which will be incubated at 37°C
4. Put the other plate in the incubator at 37°C
Results:

Anaerobic plate (put in anaerobic jar) Aerobic plate

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Exercise 4C: Testing for Enzymes Involved in Oxygen Utilization
Purpose: To test for the presence of two oxygen utilizing enzymes.
Method:
1. Grab a chunk of each culture and transfer it to 2 slides aseptically
2. On one slide place a drop of oxidase reagent – a positive result will yield a purple color within 20
seconds
3. On the other slide place a drop of catalase reagent – a positive result will result in immediate bubbling
Results:
Catalase Test:
Positive result Immediate bubbling
Negative result No bubbling

E. coli P. aeruginosa C. spyrogenes R.A. unknown K.F. unknown

Catalase + + + + +

Oxidase test: No positive results for the oxidase test were observed

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 Lab 5: Bacterial Biochemistry – Carbohydrates
Introduction: Bacteria can use a wide variety of carbohydrates which may be in the form of monosaccharides,
disaccharides, and polysaccharides. These carbohydrates are converted into glucose and used in oxidative and
fermentative pathways depending on the microorganism and the gaseous conditions it is currently in.
Exercise 5A: Using O.F. Media to Distinguish Between Oxidative and Fermentative Metabolism of a Sugar
Purpose: To learn how to use O.F. media to distinguish between oxidative and fermentative metabolism of
glucose.
Method:
1. Inoculate 2 tubes of O.F. media for each species (one will be an aerobic and the other will be an
anaerobic culture)
2. Add a small amount of sterile mineral oil to one tube which will be the anaerobic culture (1-2cm of oil)
3. Incubate tubes at appropriate temperature
Results:
Positive result for oxidative/fermentative metabolism Yellow
Negative result for oxidative/ fermentative metabolism Green

E. coli P. aeruginosa C. spyrogenes R.A. unknown K.F. unknown

Aerobic + + - - -

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 E. coli P. aeruginosa C. spyrogenes R.A. unknown K.F. unknown
Anaerobic + - - - -

Exercise 5B: Observation of Fermentation in Phenol Red Broth

Purpose: To visualize the differences in fermentation of glucose, sucrose, and lactose in Phenol Red broths.
Method:
1. Inoculate each species into one tube of each Phenol Red broth (glucose, sucrose, lactose)
2. Incubate the tubes at the appropriate temperature
Results:
Positive for acid (A) Yellow
Positive for gas (G) Bubble in the Durham tube
Nonfermentor (NF) Red, no bubble in Durham tube

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 E. coli P. aeruginosa C. spyrogenes R.A. unknown K.F. unknown
PR Glucose AG NF AG NF NF
PR Lactose AG NF NF AG AG
PR Sucrose G G G G G

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 Lab 6: Bacterial Biochemistry – Nitrogen
Introduction: Microorganisms require nitrogen to synthesize proteins and nucleic acids. It can be obtained
from many sources by hydrolyzing proteins from other organisms, by using inorganic nitrogen containing
compounds, or by using nitrogen gas from the atmosphere.
Exercise 6A: Hydrolysis of Casein on Milk Agar Plates
Purpose: To learn how microorganisms obtain nitrogen from organic sources and use it for metabolism.
Method:
1. Take a plate of milk agar and divide it into 4 pie slices
2. Streak each slice with one of the cultures to be tested
3. Incubate the plate at the appropriate temperature
Results:
Positive result for milk hydrolysis Zone of clearing surrounding growth
Negative result for milk hydrolysis No clearing

B. subtilis S. epidermidis R.A. unknown K.F. unknown

+ + + +

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Exercise 6B: Microorganism Capability to Use Lysine
Purpose: To learn how microorganisms metabolize organic nitrogen sources.
Method:
1. Inoculate each species into a tube of lysine decarboxylase media
2. Create an aerobic environment by adding mineral oil on top of the media
3. Incubate the tubes at the appropriate temperature
Results:
Positive result for lysine decarboxylation Purple
Negative result for lysine decarboxylation Yellow

B. subtilis S. epidermidis R.A. unknown K.F. unknown

- - + +

Exercise 6C: Detection of the Reduction of Nitrate to Nitrite

Purpose: To be able to determine if nitrate was reduced.
Method:
1. Week 1
a. Inoculate one tube of Tryptic nitrate broth for each species
b. Incubate tubes at appropriate temperature
2. Week 2
a. Add 1mL (about 20 drops) of reagent A and B to each tube
b. If there is no color change add a small amount of powdered zinc
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Results:
Positive result for nitrate reduction Red/purple
Negative result for nitrate reduction No color change

B. subtilis P. aeruginosa S. epidermidis R.A. unknown K.F. unknown

+ - + - -

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Positive result for reduction of nitrite Red/purple
Negative result for reduction of nitrite No color change

P. aeruginosa R.A. unknown K.F. unknown

- + +

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 Lab 7: The Kirby Bauer Method for Testing Antibiotic Susceptibility
Introduction: The effectiveness of antibiotics is not universal. Microorganisms are not always susceptible to
every antibiotic. Testing an antibiotics’ effectiveness on microorganisms in the clinical setting under strict
conditions. However, a much simpler test can be conducted for an easier and quicker answer using
supplemented Brain Heart Infusion Agar.
Purpose: To test the susceptibility of chosen bacteria using an adapted version of the Kirby-Bauer method.
Method:
1. Week 1
a. Mix the broth culture by shaking
b. Dip a sterile swab into the broth and saturate it
c. Rub the swab over the entire surface of the agar (make two plates for each species)
d. Let dry for 5 minutes
e. Obtain discs of chloramphenicol, streptomycin, tetracycline, ampicillin, penicillin, and a blank
disc
f. Take a pair of forceps out of the alcohol dish and burn off excess alcohol (repeat in between
uses)
g. Take a disc out using the forceps and press firmly to the agar
h. Place 3 discs on each plate making sure that none of the discs are too close to the edge or to each
other
i. Incubate the plates at the appropriate temperature
2. Week 2
a. Measure the diameter of the zone of inhibition of each antibiotic
Results:
Zone diameter (mm)
Antibiotic Resistant (R) Intermediate (I) Susceptible (S)
Chloramphenicol ≤12 13-17 ≥18
Streptomycin ≤11 12-14 ≥15
Tetracycline ≤14 15-18 ≥19
Ampicillin (Gram +) ≤11 12-13 ≥14
Ampicillin (Gram -) ≤20 21-28 ≥29
Penicillin Information not provided Information not provided Information not provided

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 S. salivarius (Gram +) N. subflava (Gram -)
Chloramphenicol 22 mm - S 42 mm - S
Streptomycin 8 mm - R 20 mm - S
Tetracycline 26 mm - S 36 mm - S
Ampicillin 23 mm - S 32 mm - S
Penicillin 20 mm 32 mm
Blank disc 0 mm 0 mm

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 Lab 8: Selective and Differential Medias
Introduction: Selective medias are supplemented with chemicals that inhibit the growth of certain
microorganisms. Differential medias contain chemicals that distinguish microorganisms based on
characteristics. Both media are used to identify microorganisms and medias can even be both selective and
differential.
Exercise 8A: Hemolysis on Blood Agar
Purpose: To learn how to use enriched media to differentiate microorganisms based on hemolysis.
Method:
1. Obtain 2 blood agar plates and divide each into 3 parts
2. Streak each section with one species including a swab of the back of someone’s gum line
3. Incubate the plates at the appropriate temperature
Results:
Positive result for alpha hemolysis (α) Green tint
Positive result for beta hemolysis (β) Zone of clearing
Positive result for gamma hemolysis (γ) Opaque red color

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 S. salivarius S. epidermidis S. aureus R.A. unknown K.F. unknown
γ γ β α α

Exercise 8B: Using Selective and Differential Media to Distinguish Gram-positive Organisms
Purpose: To learn how gram-positive microorganisms can be identified using selective and differential media.
Method:
1. Obtain 2 Mannitol salt agar (MSA) plates and divide each into 3 parts
2. Streak each section with one species
3. Incubate the plates at the appropriate temperature
Results:
Positive result for staphylococci Growth
Negative result for staphylococci No growth

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 S. aureus S. epidermidis E. coli R.A. unknown K.F. unknown
+ + - + +

Exercise 8C: Using Selective and Differential Media to Distinguish Gram-negative Organisms
Purpose: To learn how gram-negative microorganisms can be identified using selective and differential media.
Method:
1. Obtain 2 Eosin-methylene blue (EMB) plates and divide each into 3 parts
2. Streak each section with one species
3. Incubate the plates at the appropriate temperature
Results:
Positive for Gram-negative (G) Growth
Positive for lactose fermentation (L) Metallic blue-green sheen

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 S. typhimurium S. epidermidis S. flexerni E. coli R.A. unknown K.F. unknown
G - G GL G G

Exercise 8D: Multi-test Media

Purpose: To learn how to use multi-test media to differentiate bacteria.
Method:
1. Week 1
a. Obtain 5 slants of Triple sugar iron agar (TSIA) and 5 tubes of Sulfide indole motility (SIM)
agar
b. Inoculate 1 tube of each media with one species
i. For the SIM media simply stab the agar
ii. For the TSIA media, stab the needle down to the butt of the tube, withdraw the needle
and rub it along the surface of the slant
2. Week 2
a. For the SIM media: observe the tube for motility, then add 10-20 drops of Kovac’s reagent
b. For the TSIA media: observe the butt and slant of the tube separately
Results:
SIM Media
Positive result for reduction of sulfate to H2S (B) Black precipitate
Positive result for the presence of indole (R) Red after addition of Kovac’s
Positive result for motility (M) Growth throughout the tube

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E. coli S. flexerni R.A. unknown K.F. unknown S. tyhphimurium
R - - - B

TSIA Media
Observation (slant/butt) Symbols Interpretation
Red/yellow K/A Glucose fermented only
Yellow/yellow A/A Glucose and lactose and/or sucrose
fermented
Red/red K/K No fermentation
Yellow/yellow with bubbles A/A, G Glucose and lactose and/or sucrose
fermented, w/ gas produced
Red/yellow with bubbles K/A, G Glucose fermented only, with gas
produced
Red/yellow with bubble and black K/A, G, H2S Glucose fermented only, with gas
precipitate produced, H2S produced
Red/yellow with black precipitate K/A, H2S Glucose fermented only with H2S
produced
Yellow/yellow with black A/A, H2S Glucose and lactose and/or sucrose
precipitates produced fermented with H2S produced

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R.A. unknown S. flexerni S. typhimurium K.F. unknown E. coli
K/K K/A K/A, H2S K/K A/A, G

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 Lab 9: Differentiation of Microbial Water Contaminants
Introduction: Presumptive tests are done to detect the presence of coliform bacteria which are Gram-negative,
facultative anaerobe bacteria that ferment lactose. Coliform bacteria are usually found in the intestines of
mammals and their presence is associated with fecal contamination of water.
Exercise 9A: Using the IMViC Series to Identify Coliform Bacteria
Purpose: To learn how to use the IMViC series to differentiate Gram-negative enteric organisms.
Method:
1. Week 1
a. Obtain 6 tubes of Tryptone broth and 6 tubes of Simmon’s citrate slants and inoculate each with
one species of bacteria
b. Obtain 12 tubes of MRVP broths and inoculate two tubes for each species
c. Incubate at appropriate temperature
2. Week 2
a. Indole test: Add 10 drops of Kovac’s reagent
b. MR test: Add 4-5 drops of Methyl red pH indicator
c. VP test:
i. Add approximately 1mL (20 drops) of VP-A reagent
ii. Add approximately 0.5mL (10 drops) of VP-B reagent
iii. Gently shake the tube and let sit for 10-15 minutes
d. Citrate test: Simply observe the slant
Result:
Indole test
Positive test for tryptophanase to indole Red layer on top of liquid
Negative test for tryptophanase to indole Yellow

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 S. flexerni S. typhimurium E. aerogenes E. coli R.A. unknown K.F. unknown
- - - + - -

MR test
Positive result for mixed fermentation Red
Negative result for mixed fermentation Yellow

S. flexerni S. typhimurium E. aerogenes E. coli R.A. unknown K.F. unknown

+ + - + - -

VP test: No positive results for the VP test were observed

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Citrate test
Positive result for use of citrate as carbon source Blue
Negative result for use of citrate as carbon source Green

S. flexerni S. typhimurium E. aerogenes E. coli R.A. unknown K.F. unknown

+ + + - - -

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