Introduction to Clinical Microbiology

2nd Edition

Microbiology Lab – Fall 2009

Modified by J. Volpe

Table of Contents

Table of Contents

Course Objectives and Information:

Microbiology Laboratory Rules:

Microscope Techniques

Plating Bacteria

Sterile Technique, Spreading and Streaking Plates.

Gram Stain

Carbohydrate Fermentation (Phenol Red Broth)

Catalase Activity

Obtaining a Pure Culture

Acid Fast Stain

Environmental Affects on Bacterial Growth

Antiseptics and Antibiotics

The Enterotube

Course Objectives and Information:

  1. Demonstrate basic microbiology lab skills
  1. Demonstrate lab techniques including:
  2. Documenting accurate experimental lab observations
  3. Evaluating lab observations
  4. Write comprehensive lab reports
  5. Design and verify lab protocols

3. List the responsibilities and duties of a professional microbiology lab

Student expectations:

  1. Attend all labs.
  2. If you cannot attend your section of the lab: attending the other section is preferred with prior approval.
  3. Labs build on each other; it is crucial to keep up. If you miss a lab you are responsible for knowing the techniques in that lab because you will use them in other labs.
  4. Complete Lab Reports.
  5. Lab reports are to be typed and turned in the following lab.
  6. Lab techniques are to be completed with a lab partner. Each partner is to complete their OWN lab report.
  7. ACADEMIC HONESTY is expected! Plagiarism and academic dishonesty will result in a zero for the assignment for the first offense, an “F” for the second.

Lab Report Requirements:

-must include you AND your lab partners name on the report

-Include:

1. Introduction - describe procedure

2. Observations – document detailed observations

3. Exercises – one must answer all questions associated with each lab

4. Conclusion:

i) describe objective(s) you learned

ii) what were the difficulties in the lab technique

Lab grade consists of:

See your syllabus. There will be lab questions on exams and at least one lab essay question per exam, all assigned information from the in lab manual is testable material.

Microbiology Laboratory Rules:

General Rules

  1. Be courteous to your lab mates, class mates, and instructor. Help each other; you’ll be your best resources.
  2. You are expected to have read lab materials before coming to class, lecture materials before an exam.
  3. No talking, phone or computer use during exams. In lecture and exams, turn phones off or to vibrate.
  4. If you have a problem, speak to your instructor.

Lab Rules

  1. Report spills and accidents to your instructor.
  2. Wash your workbench top with disinfectant before and after and your hands after each lab. This is good lab procedure and P1 rules.
  3. No food or drink in the lab. No smoking, chewing gum, or chew is allowed. You must wear closed toed shoes in lab.
  4. Your gear, coats, backpacks, etc should be in the back of the lab and not at your workbench. Books, notebooks, pencils, may be at your bench.
  5. Long hair has to be tied back for fire safety reasons. No exceptions when a Bunsen burner is used anywhere in the lab!
  6. Turn off your Bunsen burner when not in use.
  7. Assume every organism is hazardous. Do not put fingers, pens, or other objects in your mouth while in the lab. Gloves are to be worn when handling known pathogens.
  8. Cultures that you are finished with need to be autoclaved and thrown away.
  1. Dispose of all materials properly. All cultures or items that have touched a culture are assumed to be hazardous and must be disposed of properly in a hazardous waste bag. If unsure, ask your instructor.
  2. Any instruments that come in contact with bacteria must be sterilized.
  3. Inoculation loops are to be flame sterilized before and after each use.
  4. Plates must be autoclaved before thrown away. Place used plates in a red biohazard bag in back of class. Plates are autoclaved in this bag and then thrown away in garbage.
  5. Glass tubes and pipettes need to be autoclaved and then washed before reuse.
  6. Slides are to be placed in a beaker with disinfectant; they will be washed and reused. Cover slips are to be discarded as broken glass or if plastic, the autoclave bag.
  7. No cultures can leave the lab for any reason.
  8. All supplies should be returned to the back of the class where you picked them up from at the beginning of lab. Lids must be securely fastened.
  9. Properly label all tubes and plates w/ your name, the date, and the name of the culture (or the experiment name).
  10. Handle all tubes and plates by gripping the tube or plate, not the lid! You also will need to wash some glassware.
  11. The slide boxes stick. When opening, put a slide box on a flat surfce & carefully pry open the lid. Otherwise all the slides may fly out and break on the floor.
  12. Stains used will stain clothes, lab benches, skin. Contain mess by covering lab benches with paper towel or lab bench cover, wear gloves, and work over a sink or large beaker.
  13. Clean, carry, and maintain your microscope in the proper fashion.
  14. Immersion oil is to be cleaned off the oil immersion lens after each use; this is to be done with a piece of lens paper. DO NOT USE A KIMWIPE or PAPERTOWEL!!
  1. Do not deviate from a taught technique w/o permission from the instructor. You will be taught techniques to minimize contamination of yourself and the surrounding environment as well as sterile techniques.

Microscope Techniques

Objective:

  1. Identify components of the light microscope and demonstrate their functions
  2. Identify components and demonstrate function of fluorescence microscopy and electron microscopy

Resources:Your textbook

Supplemented by lecture and lab notes

Supplies:

□light microscope

□slide

□water bottle

□lens paper

□oil immersion oil

□ethanol

□kimwipe

□scissors

Lab procedure:

  1. Cut a lower case “e” from the newspaper.
  1. Take a clean slide. If slide is not clean wipe with a kimwipe and a drop of ethanol.
  1. Mount the “e” to the slide by placing a drop of water, from a water bottle, on the slide and place “e” on top. Make sure that the “e” is not moving around. Note which way the “e” is facing.
  1. Starting with the lowest setting focus the microscope and work to higher settings until the entire “e” is visible. Draw the “e” in lab report viewed under the microscope and in plain sight.

NOTE: be sure the provide magnification below and label

  1. Take a fixed slide from front of class. Work with the microscope to get a clear view of the culture. Try moving all the functional components of the microscope to see what makes a clear image.

The microscope will be adjusted differently for everyone’s eyes.

a)All microscopes in the lab have two eye pieces; you should use both of them. It is common to want to close one eye when starting to use a microscope, concentrate on using both; adjust the width to make the lenses fit the diameter of YOUR face.

b)Change the light intensity to get the clearest view and best colour illumination.

c)The diaphragm can concentrate light.

  1. Starting with lowest magnification, work up until using oil immersion lens. Draw in lab report.
  1. Repeat this process with 3 fixed slides. Drawing each in your lab report.
  1. After using oil immersion the lens MUST be cleaned by wiping off immersion oil with a lens paper. DO NOT USE A KIMWIPE!!!!

Exercises:

  1. Fill in blanks in on microscope image. For components that are adjusted, write a brief description of what happened to the image when you adjusted it.
  1. What did you notice about the plain view “e” and the microscope image of the “e”?
  1. What are the lowest magnification and the highest magnification on the microscopes in lab?
  1. When adjusting the microscope from 400X to 1000X you should use the course focus adjustment, True or False?
  1. To get a dark field how would you adjust the microscope?
  1. When is it best to use dark field microscopy?
  1. Describe a possible usage of fluorescence microscopy.
  1. EM is particularly useful is imaging of what type of pathogen? Why?
  1. When putting microscope away; in what position (magnification/stage/cord/cover) should the microscope be in?
  1. Explain the importance of using lens paper.

Plating Bacteria

Objective:

  1. List the general ingredients and the major differences between agar, media, and broth.
  2. List when you would use agar, media, and broth.
  3. Demonstrate pouring agar on a plate / tube or slants using sterile techniques.

Supplies needed:

□LB or TSA

□E. Flask

□Weigh boats

□Spatula

□dH20

□plates

□tubes

□tube racks

□stir bar/rod

□gloves

□graduated cylinders

□Bunsen burner/striker

Procedure for making agar plates:

  1. Calculate how much agar you will need to make 250ml of each. You are only responsible to make one type of agar, assigned in class.
  2. LB: 35g/L
  3. TSA: 40g/L
  1. Weigh agar: using a spatula scoop dehydrated agar out of container onto weigh boat on scale.

***Do not take agar that has been out of the container and put it back into the container, this is not done to keep the stock container non contaminated. This rule should be followed when measuring anything from the lab stock.

  1. Measure 250mL H20; half of the class will make LB and half will make TSA. Assigned day of lab.
  1. Combine agar with H20 in an Erlenmeyer flask.

***Best if you put agar into the flask first and then H2O because the agar will stick to the sides of the flask where the water is, making it more difficult to mix all the agar into solution.

  1. Boil to get agar into solution. Place flask on hot plate starting with heat at about 5 on a 10 scale to start. Every hot plate is slightly different so it is best to start at a lower temperature and increase. When available use a stir bar to mix. In this lab we will use a stir rod to make sure agar is mixed into solution.

***Do not leave hot plate unattended when turned on.

  1. Autoclave: 15min 121oC
  2. we will autoclave all at once so wait for the rest of the class if completed early.
  3. Autoclave demonstration will be done.
  1. Allow to cool to 56oC
  2. This can be done using a water bath or by leaving at room temperature.
  1. Pouring:
  2. Plates: lift lid at an angle above plate with less dominant hand. With dominant hand pour agar solution to cover plate
  3. Tubes: fill tube 1/3 full with agar solution
  4. Slants: fill tube 1/3 full with agar solution. Prop test tube rack so that agar hardens at a slant
  1. Allow to cool and harden.
  2. Invert plates and stack.
  1. Place lids on tubes.
  2. Tubes can be used as tubes or agar can be heated up again and poured into plates.
  1. Refrigerate 4oC for storage

Exercises:

  1. Write step-by-step protocol for making an agar plate. Include details.
  2. How much dehydrated agar (LB and TSA) was needed to make 250ml of agar? Show calculation.
  3. When would you add antibiotics, if you wanted an ampicillin slant?
  4. What is the advantage of a plate, tube, slant?

Sterile Technique, Spreading and Streaking Plates.

Objective:

  1. List how to control contamination
  2. List why contamination control is important
  3. Perform techniques to prevent contamination
  4. Perform spreading and streaking techniques
  5. List proper way to clean up after a microbiology lab and why its important

Resources:Textbook chapters.

Supplemented by lecture and lab notes

Supplies:

□4 different broth bacteria

□dropper/bulb

□beaker

□L rod

□Inoculation loop

□Ethanol

□Sharpies

□Bunsen burner/striker

□Plates (from 2a)

Procedure:

Spreading:

  1. Pipet 1ml bacteria on agar: You will use your plates you made in 2a, broth bacteria cultures will be provided at front of class. For this lab quantity is not important therefore we will use a dropper and bulb to transfer bacteria. After use both should be placed in 10% Lysol solution.
  1. Dip L-rod in ethanol and flame L-rod. Flaming is a way of sterilizing instruments; the flame of the Bunsen burner kills the bacteria that are on the instrument. This is the same sequence used to flame an inoculation loop or needle.

NOTE: Make sure to hold rod at an angle so that ethanol does not run towards your hand.

  1. Allow L rod to cool. If you do not allow your L rod to cool the heat can kill the bacteria on the plate.
  1. Spread bacteria. Using L rod distribute bacteria evenly across surface of agar.
  2. put in incubator for 24-48 hours
  1. Draw and record you observations.

Plate Streaking (Colony Streaking -Quadrant Method):

  1. On the bottom of an agar plate divide the plate into 4 quadrants. Write your initials and date. Label each quadrant with bacteria name that you will be streaking that quadrant with. Leaving one quadrant labeled control.
  1. flame sterilize inoculation loop. Same procedure as L rod but your can cool loop by placing loop in on an agar plate. The loop will cause a ring in the agar plate.
  1. streak one quadrant of plate with one bacteria.
  1. flame and repeat with next bacteria
  1. leave 1 quadrant not streaked as a control
  1. put in incubator for 24-48 hours
  1. Draw and record observations

Exercises:

  1. Why is it important to use sterile technique?
  1. Media, broth and agar are sterilized using different techniques. What are they and why are they different?
  1. Give 2 reasons why wearing gloves in lab is important.
  1. Name 5 body sites that are normally sterile and 5 body sites that are not. Why is it important to know this information?
  1. What should you do when finished with a plate, tube or instrument that has come in contact with pathogen?
  1. What would be the function of adding antibiotics/antifungal regents to broth or agar?
  1. When viewing your plate after incubation; if you notice growth the control quadrant what would you assume?
  1. A colony on an agar plate is a group of cells all from one cell plated during streaking. True or False? (One colony = one cell)

Gram Stain

Objective:

  1. List and demonstrate the 4 step gram stain procedure
  2. Draw and label the bacteria cell wall

Resources: Lecture and lab notes

Supplies:

□Bacteria (gram negative and gram positive)

□Water bottle

□Slides

□Lens paper

□Immersion oil

□Inoculation loop

□Beaker 100ml/500ml

□Ethanol

□Iodine

□Crystal Violet

□Red Safrinin

□Kimwipe

□Microscope

□Bunsen burner/striker

Observation:

***View under microscope, draw, record magnification, identify bacteria shape and its growth pattern or patterns***

  1. Look at 4 pre-prepared slides. (Record slide title.)
  1. Perform 3 successful gram stains; one gram negative, one gram positive, a mixture.

It is best to start with the gram negative bacteria. This is best because the decolourizing step can be tricky. If you start with gram negative you can gain an understanding of how long you need to decolourize for.

NOTE: If you do not get what you expected redo until you get it right.

  1. Perform 1 gram stain with unknown bacteria.

Procedure:

Fixing bacteria to a slide:

Broth culture:

-transfer a very small volume of broth containing bacteria to slide. This can be done with a with a flame sterilized inoculation loop.

-swipe slide through flame of Bunsen burner.

Bacteria from agar surface:

-place a small drop of water on a slide

-using a sterile inoculation loop scrap bacteria off agar onto inoculation loop.

-mix bacteria with drop of water of slide.

-swipe bacteria through flame of a Bunsen burner.

these steps are the most crucial of the entire gram stain:

-Too much bacteria will make viewing difficult because bacteria will be too close together.

-Not fixing the bacteria well enough will cause them to be washed away during gram stain.

-swiping slide through Bunsen burner too much will burn bacteria and kill it.

***swiping slide through Bunsen burner is done to evaporate this liquid. If liquid at anytime bubbles, your bacteria are dead. This step takes patience***

 put your slide with fixed bacteria balancing across a 400ml beaker with a lab bench cover underneath.

  1. Apply 1-2 drops crystal violet stain for 60secs.
  2. Rinse with water to remove excess stain
  1. Apply 1-2 drops iodine 30 secs
  2. Rinse with water
  1. Decolourize with 70% ethanol until run off is clear. Gram positive gram will appear purple after this step and gram negative will be colourless, therefore don’t decolourize too long or you may get a false result.
  1. Counter stain with Safrinin 1-2 drops for 60secs
  2. Rinse with water
  3. Blot with Kimwipe (do not wipe!!!)

-using the microscope find a view where you can see bacteria shape and colour. (start with low power working your way to oil immersion)

  1. Place slides in 10% Lysol solution. Inoculation loops are to be flame sterilized at the end of lab. Throw bench covers in garbage.

Exercises:

  1. Gram negative is what colour? Gram positive is what colour?
  1. What is the function of:
  2. Crystal violet:
  3. Iodine:
  4. 70% ethanol:
  5. Safrinin:
  1. What shape is: Draw each.
  2. Cocci:
  3. Bacilli:
  4. Spirochete
  5. Strepto
  6. Staphylo
  7. Diplo

Carbohydrate Fermentation (Phenol Red Broth)

Objective:

  1. List the biochemical process of fermentation
  2. Accurately describe fermentation and its uses in microbiology.

Resources: textbook Supplemented by lecture and lab notes

Supplies:

□5 TSA Durham tubes w/ phenol red

□Bacteria:

  • E. Coli
  • Alcaligenes faecalis
  • Salmonella typhimurium
  • Staphylococcus aureus

□Bunsen burner/striker

□Inoculation loop

□Ethanol

□Beaker

□Test tube rack