LAB NOTES FOR EXAM 1

EX. 2-1: DIVERSITY AND UBIQUITY OF MICROORGANISMS

Purpose:

• To demonstrate the omnipresence of microorganisms in the environment.
• To culture microorganisms by collecting samples using a swab and transferring them to an agar plate.

Media & Materials:

• 1 Tryptic Soy Agar (TSA) plate
• 1 sterile swab
• 1 tube of sterile saline per person

Procedure:

1. Sample Collection:

   • Use different methods for sample collection; confer with lab partners to ensure diversity in sources.
   • Each table should have 4 plates (5 if there is an extra person).

2. Labeling the Plate:

   • Label the TSA plate with your name, date, and source of inoculum (e.g., desktop, doorknob, skin).
   • Public body surfaces can be used, except for the mouth.

3. Inoculation:

   • Transfer microbes by swabbing the surface of the gathering area and gently rubbing the swab on the agar plate.
   • Follow the instructor’s demonstration for spreading cells.

4. Incubation:

   • Incubate plates at 37^ ext{o}C until the next lab period.

5. Observation:

   • During the next lab meeting, observe plate colonies and note colors, textures, and shapes on your data sheet.
   • The individual growth areas are referred to as colonies, consisting of millions of identical cells from a single parent cell.

6. Disposal:

   • Dispose of cultures in Biohazard waste containers after observations.

EX. 1-3: ASEPTIC TRANSFER AND INOCULATION TECHNIQUES

Purpose:

• To learn proper handling and transfer of bacterial cultures to prevent contamination.

Cultures:

• Broth culture of Serratia marcescens and agar slant culture of Serratia marcescens.

Media:

• 1 Tryptic Soy Agar (TSA) slant
• 1 Tryptic Soy Broth (TSB) per student

Procedure:

1. Labeling:

   • Label all sterile media tubes with organism name, date, and initials before inoculating.

2. Inoculation Protocol:

   • Use Serratia marcescens, which produces a red pigment under appropriate conditions for identification of successful transfers.
   • No pink/red should appear in freshly inoculated tubes during the current lab.

3. Transfer between Cultures:

   • Inoculate slant using the broth culture and vice versa.

4. Safety Protocol:

   • Replace the screw top on tubes and back off about 1/2 turn for air circulation before incubation.

5. Incubation:

   • Incubate all cultures at 37^ ext{o}C until the next lab.

6. Observation:

   • Observe cultures during the next lab meeting and dispose of them accordingly.

EX. 1-4: STREAK PLATE ISOLATION OF PURE CULTURE (PART 1)

Purpose:

• To isolate single colonies to cultivate a pure culture.

Cultures:

• Mixed broth culture of Serratia marcescens and Staphylococcus aureus.
• Mixed broth culture of Escherichia coli and Micrococcus luteus.

Media:

• 2 TSA plates per pair of students

Procedure:

1. Inoculation:

   • Inoculate separate agar plates with mixed broth cultures following the streak plate procedure as per the lab manual.

2. Technique:

   • Ensure to streak gently to avoid digging into the agar, which could hinder isolated growth.

3. Incubation:

   • Incubate plates upside down (lid on the bottom) in a wire basket at 37^ ext{o}C until the next lab period.

4. Observation:

   • During the next lab meeting, analyze plates for visible differences in colony morphology and record on data sheets.
   • Identify colony characteristics: Serratia marcescens (pink or reddish), Staphylococcus aureus (small white), Escherichia coli (beige colonies 2-3 mm), and Micrococcus luteus (smaller yellow colonies).

EX. 1-4 (AND EX. 2-2): STREAK PLATE ISOLATION OF PURE CULTURE (PART 2)

Purpose:

• Isolate a pure culture from mixed culture plates by restreaking from an isolated colony.

Media:

• 4 TSA plates per pair of students

Procedure:

1. Label Plates:

   • Label fresh plates with names of the organisms from mixed cultures, initials, and date.

2. Aseptic Touch:

   • Aldasely touch the sterile loop to the selected colony to transfer without visible inoculum to ensure purity.

3. Restreaking:

   • Use a streak plate dilution method to inoculate fresh plates.
   • Remember to flame the loop between quadrants.

4. Incubation:

   • Invert and incubate the plates at 37^ ext{o}C until the next lab.

5. Observation:

   • Use these plates for Ex. 2-2: Colony Morphology; record data and observations.

EX. 2-2: COLONY MORPHOLOGY

Purpose:

• Observe differences in colony morphology between species grown on solid media.

Organisms:

• Broth cultures of Bacillus subtilis and Mycobacterium smegmatis.

Media:

• Two TSA plates per pair

Procedure:

1. Inoculate Plates:

   • Use a sterile loop to inoculate the respective organisms onto separate TSA plates using streak plate method, avoiding cross-contamination especially with Bacillus species.

2. Incubation:

   • Incubate all plates at 37^ ext{o}C until the next lab; monitor for growth consistency. Mycobacterium and Micrococcus may require extended incubation time.

3. Observation:

   • Document observations using proper terminology and reference any demonstration plates of other organisms present in the lab.

4. Disposal of Cultures:

   • Cultures must be disposed of in red autoclave bags placed in large gray Biohazard waste containers.

EX. 2-3: GROWTH PATTERNS ON SLANTS

Purpose:

• Observe variations in morphology across different bacterial species grown on slant media.

Organisms:

• Bacillus subtilis and Mycobacterium smegmatis
• Mixed culture plates from Ex. 1-4.

Media:

• 6 TSA slants

Procedure:

1. Label Slants:

   • Label each slant tube with date, organism name, and initials.

2. Inoculation:

   • Inoculate each tube using aseptic techniques. For some organisms, you may need to use pure culture plates as needed.

3. Observation:

   • During the next lab, record observations for all slants using classroom vocabulary and concepts.

4. Control Slant:

   • Use a fresh TSA slant as uninoculated control but return after the observations.

EX. 2-4: GROWTH PATTERNS IN BROTH

Purpose:

• Observe differences in morphology among various bacterial species growing in broth media.

Organisms:

• Bacillus subtilis and Mycobacterium smegmatis
• Mixed culture plates from Ex. 1-4.

Media:

• 6 Tryptic Soy Broth (TSB) tubes

Procedure:

1. Label Broth Tubes:

   • Label tubes with date, organism name, and initials. Set up for the same cultures as in Ex. 2-3.

2. Inoculation:

   • Carefully transfer organisms from mixed cultures into the broth tubes.

3. Observation:

   • Document observations regarding growth characteristics in your data sheets during the next lab period.

EX. 2-7: FLUID THIOGLYCOLLATE: ATMOSPHERIC OXYGEN REQUIREMENTS

Purpose:

• Observe growth patterns of organisms according to oxygen needs.

Organisms:

• Broth cultures of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Neisseria sicca, and Clostridium butyricum.

Media:

• Thioglycollate broths and one supplemented thioglycollate.

Procedure:

1. Labeling Tubes:

   • Prepare labels for each organism, ensuring clarity, and remember to use initials and date.

2. Inoculation:

   • Use a sterile disposable bulb pipette to transfer 0.25 ml of each culture into the respective broth without introducing air bubbles.

3. Disposal:

   • Properly discard the used pipette in the autoclave collection.

4. Incubation:

   • Incubate tubes at 37^ ext{o}C until the next lab without shaking or disturbing them before observations.

5. Observation:

   • Make assessments on growth patterns related to oxygen requirements and record data in your observation sheets.

EX. 7-3: THE KIRBY-BAUER ANTIBIOTIC SENSITIVITY TEST PROCEDURE

Purpose:

• Observe the effects of various antibiotics on Gram-positive and Gram-negative organisms.

Organisms:

• Broth cultures of Escherichia coli and Staphylococcus aureus.

Media and Materials:

• 2 tubes sterile TSB
• 2 disposable 1 ml bulb pipettes
• 2 large Mueller-Hinton agar plates
• 2 sterile cotton swabs
• Antibiotic disks (streptomycin, tetracycline, penicillin, chloramphenicol, cephalothin, erythromycin, novobiocin, vancomycin)

Procedure:

1. Dilution Preparation:

   • Dilute each stock culture 1:50 by combining 0.25 ml culture with 5 ml sterile TSB and label appropriately.

2. Plating:

   • Use sterile swabs to inoculate plates, ensuring even coverage by rotating the swab in two perpendicular directions.

3. Disk Placement:

   • Use flamed forceps to place antibiotic paper disks on agar, ensuring they do not penetrate the agar’s surface.
   • No need to label the plates with antibiotic names as each disk already has an identifier.

Interpretation of Results:

• Measure diameter of the zone of inhibition to determine bacterial sensitivity:
  • If the size is smaller than the indicated range, indicate resistance; larger sizes indicate susceptibility.

EX. 2-9: TEMPERATURE

Purpose:

• Determine the optimum growth temperatures for different bacterial species.

Organisms:

• Escherichia coli, Staphylococcus aureus, Bacillus stearothermophilus, Pseudomonas fluorescens

Media:

• Four TSA plates per pair

Procedure:

1. Preparation of Plates:

   • Label sections of each plate for temperatures 4°C, 25°C, 40°C, and 60°C, along with names of organisms.
   • Make straight line inoculations in each temperature sector.

2. Incubation:

   • Place plates in appropriate temperature baskets for a duration until the next lab session.

3. Observation:

   • Record growth levels using a scale (0-3). Assess organism types: psychrophiles, mesophiles, or thermophiles, and discuss their likely environments.

EX. 2-11: OSMOTIC PRESSURE

Purpose:

• Demonstrate the impact of osmotic pressure on microbial growth.

Organisms:

• Escherichia coli, Staphylococcus aureus, Bacillus subtilis.

Media:

• 5 beef heart infusion (BHI) plates with varying salt concentrations: 0.85%, 5%, 7.5%, 10%, and 20% NaCl.

Procedure:

1. Labeling:

   • Label each sector of the plate with organisms and your identifiers.

2. Inoculation:

   • Use straight line inoculations for each organism on all five plates and incubate at 37^ ext{o}C until next lab.

3. Observation:

   • Evaluate plates for growth; record salt concentration data supporting growth and identify organisms as halophiles or others as relevant.

EX. 6-1: STANDARD PLATE COUNT

Purpose:

• Determine cell concentration of a concentrated broth culture through serial dilution and spread plate techniques.

Organisms:

• Escherichia coli.

Media:

• 4 TSA plates, 4 tubes of 4.5 mL saline solution.

Procedure:

1. Labeling:

   • Label TSA plates with dilution ratios: 1/100, 1/1,000, 1/10,000, and 1/100,000; do the same for saline tubes.

2. Preparing Dilutions:

   • Use a pre-diluted culture at 1/10, add 0.5 mL into the 1/100 and continue serial dilution.

3. Spreading:

   • Transfer 0.1 mL of each diluted culture to the corresponding TSA plates, evenly distributing with a spreader.

4. Incubation:

   • Tape plates and incubate upside-down at 37^ ext{o}C until the next lab session.

5. Counting Colonies:

   • Count colonies on each plate and calculate the original culture’s concentration using the formula:
     ext{Concentration} = rac{ ext{# colonies} imes ext{dilution factor}}{ ext{volume plated}}
   • Example: If 25 colonies appear on a plate from a dilution factor of 100,000 and 0.1 mL plated, the calculation reflects a concentration of rac{(25 imes 100,000)}{0.1} = 25,000,000 ext{ cells/mL}