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Microbiology Lab Notebook: Microscopy & Mounting Techniques, Exams of Nursing

A.T. Still University of Health Sciences (ATSU)Nursing

A comprehensive guide to keeping a laboratory notebook in microbiology, covering essential aspects like sterilization techniques, microscopy, and staining methods. It includes detailed instructions on using an autoclave, operating a light microscope, and performing various staining techniques like gram staining and acid-fast staining. The document also features numerous exercises and questions to reinforce understanding and promote critical thinking.

Typology: Exams

2023/2024

Available from 10/29/2024

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TK01 Keeping a Lab Notebook

Microbiology Lab1

Course Objectives:

Cultivation of samples (growth conditions) - equipment used

Identification of samples (biochemical assays) - tests available

Evaluation of samples (microscopy) - visualizations and recognitions key concepts

Basic Equipment:

1. Cleaning: autoclave 125 ° C, steamed, heat and pressure sterilization, chamber with thick door,

not dry air but steamed - more efficient, make sure depressurized before opening

Time restrictions: no autoclave then use steam (minutes) vs. hot air (hours)

2. Growing: fixed incubator = 37 ° C; shaker incubator = 37 ° C, for growth mediator, rotates and shakes

in circular motions to aerate and help optimize bacterial growth

3. Visualizing: Microscopy, can't see with just the naked eye, must know parts of microscope

4. Storing: Refrigerate at 4 ° C, slows bacterial growth, prolongs life of sample at this

temperature, when not finished with experimentation

Lab Safety:

1. Never eat or drink in lab - contamination risks

2. Wear Personal Protective Equipment (PPE):

Use gloves: latex, nitrile (hypoallergenic) purple,blue or green, cold thermal - liquid

nitrogen, etc., heat thermal resistant gloves - extreme hot conditions

Eyewear protection - goggles

Lab coat - dependent on working with materials that may be spilt on clothes

3. NEVER leave lab while wearing PPE includes: Public Hallway, Restroom, Cafeteria

Lab Notebook:

First Name Initial, Last name initial, Experiment #, Title of Experiment

Example: TK01 Keeping a Lab Notebook

Discover Exams of Nursing A.T. Still University of Health Sciences (ATSU)

Partial preview of the text

Download Microbiology Lab Notebook: Microscopy & Mounting Techniques and more Exams Nursing in PDF only on Docsity!

TK01 Keeping a Lab Notebook

Microbiology Lab

Course Objectives:

Cultivation of samples (growth conditions) - equipment used

Identification of samples (biochemical assays) - tests available

Evaluation of samples (microscopy) - visualizations and recognitions key concepts

Basic Equipment:

1. Cleaning: autoclave 125 ° C, steamed, heat and pressure sterilization, chamber with thickdoor,

not dry air but steamed - more efficient, make sure depressurized before opening

Time restrictions: no autoclave then use steam (minutes) vs. hot air (hours)

2. Growing: fixed incubator = 37 ° C; shaker incubator = 37 ° C, for growth mediator, rotates andshakes

in circular motions to aerate and help optimize bacterial growth

3. Visualizing: Microscopy, can't see with just the naked eye, must know parts of microscope

4. Storing: Refrigerate at 4 ° C, slows bacterial growth, prolongs life of sample at this

temperature, when not finished with experimentation

Lab Safety:

1. Never eat or drink in lab - contamination risks

2. Wear Personal Protective Equipment (PPE):

Use gloves: latex, nitrile (hypoallergenic) purple,blue or green, cold thermal - liquid

nitrogen, etc., heat thermal resistant gloves - extreme hot conditions

Eyewear protection - goggles

Lab coat - dependent on working with materials that may be spilt on clothes

3. NEVER leave lab while wearing PPE includes: Public Hallway, Restroom, Cafeteria

Lab Notebook:

First Name Initial, Last name initial, Experiment #, Title of Experiment

Example: TK01 Keeping a Lab Notebook

Objective: Developing an organized lab notebook for documenting experiments, data,

procedures and records.

Answer the following questions.

At what temperature is the fixed incubator set to, as presented in the lab video?

37°C

At what temperature should you refrigerate bacterial samples? Explain why this is beneficial.

4°C. This temperature slows bacterial growth and prolongs the life of the sample.

What are the FOUR types of gloves presented in the lab video?

Latex, Nitrile, Thermal cold, Thermal heat

What THREE rules were discussed in regards to lab safety that would protect you and others from contamination? **1. Never eat or drink in the lab
Always wear appropriate PPE
Never leave the lab wearing PPE**
What are the main sections that should be found in a lab notebook? Name at least 4.

Objective, Procedure, Notes, Results and Deviations

Answer the following questions.

stage clips

coarse/ fine focus - inner knob

iris diaphragm - light enters microscope

light source - standard halogen bulb

base - microscope sits, hold onto this part while moving

Visualization:

Types of objects: Dry vs. oil - give better resolution and clear magnification, better images

qualities

Intensity of Light source: Too bright = saturation; too dark = low visibility

Power of Objective x Power of Eyepiece = Total Magnification

Cell diameter = 15 mm, using a 40 X objective, a 5X eyepiece, then cell appears to the eye 200

times larger (40 times 5 = 200X) or approximately 3,000 mm in diameter

Begins with higher magnification for tissue/bacteria samples, if not sure then begin w lowest

magnification and work way up.

Seeing the sample - adjust stage, avoid hitting the glass side, should see a single circle,

overlapped, if too dark or too light then needs adjusting until you see the sample clearly.

Some microscopes use arrows to center and find sample clearly.

Identify the part of the microscope indicated by the arrow.

Neck (or arm)

Identify the part of the microscope indicated by the arrow.

Results: Magnification = objective X eyepiece

As you looking through the microscope you wish to dim (or limit) the amount of light entering into the eyepiece—what component of the microscope other than the light source itself can be adjusted to make these modifications?

Diaphragm

Answer the following questions

What objective power is best suited if you are uncertain what the sample is and where to begin?

4x (or lowest power objective)

TK03 Mounting Techniques

Objective: Microscopic examination of bacterial samples through various staining

techniques.Identify color and shape of given samples.

Procedure(s):

Dry Mount

1. Clean slide (ethanol 70%)

2. Circle area on slide for easy location of specimen (optional)

3. Apply organism to slide:

From a culture: use sterile loop to spread onto slide (optional)
From plate: use sterile loop to pick colonies and mix with a drop of distilled water

4. Air dry at room temperature until all moisture has evaporated

Procedure(s):

Wet Mount

1. Clean slide (ethanol 70%)

2. Circle area on slide with wax / hydrophobic pen

3. Apply organism to slide:

From a culture: use sterile loop to spread onto slide
From plate: use sterile loop to pick colonies and mix with a drop of distilled water

4. View under microscope

a. Wet Mount is ideal for viewing the motility of an organism. Do not dry out.

Procedure(s):

Gram Staining

Mount

1. Clean slide (ethanol 70%)

You are viewing a sample of bacteria that is 3 mm in diameter through a 40x objective lens. The eyepiece has a magnification power of 10x. What size will the sample appear through the eyepiece?

1200 mm in diameter (or 400x’s larger)

Based on the microscope shown in the lab lecture, which objectives would NOT require placing oil on the slide?

4x, 10x and 40x

Acid Fast Staining:

- Strong resistance to decolorization

- Very few structures are acid fast

- Commonly used to identify mycobacterium

- Carbolfuchsin dye retained (red dye)

Negative Staining:

- Commonly used to identify organisms with opaque structures

- Dark background via nigrosin dye

- Negatively charged, repelled from membrane

Results:

Staphylococcus Aureus = purple background, round (gram +)

Escherichia Coli = pink, shape rod (gram - )

Bacillus Subtilis = dark purple (gram +)

Lab 03 Answer the following questions.

List the 4 main steps used to prepare a WET mount and indicate which (if any) step is optional.

1 – Clean slide

2 – Circle area on slide with wax/hydrophobic pen

3 – Apply organism to slide

4 – View under microscope

*Note: there are no optional steps in the wet mount.

When preparing a glass slide for a dry mount, what cleaning solution was used?

70% ethanol

You wish to study the motility of a particular bacterium. What type of mounting technique would you use? Explain youranswer.

Safranin

Answer the following questions.

Why do Gram-negative bacteria stain red?

Gram-negative bacteria have a thin peptidoglycan layer in their cell wall. During the alcohol rinse step, the cell wall cannot retain the crystal violet dye. It will be washed away. The cell wall will then absorb the counter stain, safranin.

Identify the stain and shape of the bacteria pictured below.

Gram-positive; Cocci (spherical)

When might you want to utilize a negative stain technique in the lab? What is the name of the primary dye used in this technique?

A negative stain is used to identify organisms with an opaque structure. Nigrosin dye.

Why do bacteria repel the dye nigrosin?

TK04 Growth Media

Objective: Understanding types/uses of growth media for isolation and identifying unknown

bacterial samples.

Procedures:

4 - Phase dilution streaking: Clonal Isolation- purpose is to

isolate individual bacterial colonies

1. Use sterile loop spread culture in area

2. Use NEW sterile loop drag through end of area #1 ONCE

3. Use NEW sterile loop drag through end of area #2 ONCE

4. Use NEW sterile loop drag through end of area #3 ONCE

* Use the back and forth motion to dilute culture in each zone.

* Invert plate & incubate at 37 degrees Celsius overnight.

[Non - selective agar]

All materials must be place in a biohazardous waste bag and placed into an autoclave for sterilization.

What are the Gram status, shape and identification of organism #1 from the Gram stain procedure?

Gram-positive; Cocci (spherical) clusters; Staph aureus

What are the Gram status, shape and identification of organism #4 from the Gram stain procedure?

Microbiology Lab Notebook: Microscopy & Mounting Techniques, Exams of Nursing

Related documents

Partial preview of the text

Download Microbiology Lab Notebook: Microscopy & Mounting Techniques and more Exams Nursing in PDF only on Docsity!

TK01 Keeping a Lab Notebook

Microbiology Lab

Course Objectives:

Cultivation of samples (growth conditions) - equipment used

Identification of samples (biochemical assays) - tests available

Evaluation of samples (microscopy) - visualizations and recognitions key concepts

Basic Equipment:

1. Cleaning: autoclave 125 ° C, steamed, heat and pressure sterilization, chamber with thickdoor,

not dry air but steamed - more efficient, make sure depressurized before opening

Time restrictions: no autoclave then use steam (minutes) vs. hot air (hours)

2. Growing: fixed incubator = 37 ° C; shaker incubator = 37 ° C, for growth mediator, rotates andshakes

in circular motions to aerate and help optimize bacterial growth

3. Visualizing: Microscopy, can't see with just the naked eye, must know parts of microscope

4. Storing: Refrigerate at 4 ° C, slows bacterial growth, prolongs life of sample at this

temperature, when not finished with experimentation

Lab Safety:

1. Never eat or drink in lab - contamination risks

2. Wear Personal Protective Equipment (PPE):

Use gloves: latex, nitrile (hypoallergenic) purple,blue or green, cold thermal - liquid

nitrogen, etc., heat thermal resistant gloves - extreme hot conditions

Eyewear protection - goggles

Lab coat - dependent on working with materials that may be spilt on clothes

3. NEVER leave lab while wearing PPE includes: Public Hallway, Restroom, Cafeteria

Lab Notebook:

First Name Initial, Last name initial, Experiment #, Title of Experiment

Example: TK01 Keeping a Lab Notebook

Objective: Developing an organized lab notebook for documenting experiments, data,

procedures and records.

37°C

stage clips

coarse/ fine focus - inner knob

iris diaphragm - light enters microscope

light source - standard halogen bulb

base - microscope sits, hold onto this part while moving

Visualization:

Types of objects: Dry vs. oil - give better resolution and clear magnification, better images

qualities

Intensity of Light source: Too bright = saturation; too dark = low visibility

Power of Objective x Power of Eyepiece = Total Magnification

Cell diameter = 15 mm, using a 40 X objective, a 5X eyepiece, then cell appears to the eye 200

times larger (40 times 5 = 200X) or approximately 3,000 mm in diameter

Begins with higher magnification for tissue/bacteria samples, if not sure then begin w lowest

magnification and work way up.

Seeing the sample - adjust stage, avoid hitting the glass side, should see a single circle,

overlapped, if too dark or too light then needs adjusting until you see the sample clearly.

Some microscopes use arrows to center and find sample clearly.

Results: Magnification = objective X eyepiece

TK03 Mounting Techniques

Objective: Microscopic examination of bacterial samples through various staining

techniques.Identify color and shape of given samples.

Procedure(s):

Dry Mount

1. Clean slide (ethanol 70%)

2. Circle area on slide for easy location of specimen (optional)

3. Apply organism to slide:

4. Air dry at room temperature until all moisture has evaporated

Procedure(s):

Wet Mount

1. Clean slide (ethanol 70%)

2. Circle area on slide with wax / hydrophobic pen

3. Apply organism to slide:

4. View under microscope

a. Wet Mount is ideal for viewing the motility of an organism. Do not dry out.

Procedure(s):

Gram Staining

Mount

1. Clean slide (ethanol 70%)

Acid Fast Staining:

- Strong resistance to decolorization

- Very few structures are acid fast

- Commonly used to identify mycobacterium

- Carbolfuchsin dye retained (red dye)

Negative Staining:

- Commonly used to identify organisms with opaque structures

- Dark background via nigrosin dye

- Negatively charged, repelled from membrane