BIO 240 Lab Manual

Spring 2025

Joliet Junior College

M. Lee and L. Crump (original authors)
C. Dobbs, L. Smithson (editors, contributors)

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 General Laboratory Instructions

1. Preparing for Lab: Refer to the JJC Biology Laboratory Safety document you received in lab. Read
your assigned lab exercise before coming to class. Assigned activities are listed on the syllabus.
Prepare a checklist of procedures before coming to class, noting any special instructions or changes
described by your instructor. Complete the review questions and lab report section as you complete
each lab.

2. Never eat or drink in the laboratory. Avoid putting objects (pencils, pens, etc.) in your mouth at all
times. Do not apply make-up or lotions while in the lab. Do not use your cell phone while in the lab!
Do not place personal effects (book bags, coats, etc.) on the lab bench. The possibility of microbial or
chemical contamination must always be considered. Though the bacteria used in this lab are relatively
safe, all should be considered pathogenic.

3. During the Lab Period.: Using the disinfectant provided to disinfect your work area before and after
each lab period. Dry the area with the paper towels provided. This paper must be disposed in the
trashcans near your work area. Wash your hands before and after each lab period.

4. If you spill a microbial culture, notify your instructor immediately. Your instructor will assist you
to cover the spill with paper towels and pour laboratory disinfectant over both. Wait 15 minutes, and
then discard all contaminated materials in their appropriate containers.

5. After the Lab Period. Please follow these guidelines for disposal of materials:
• Place all unbroken glass culture tubes in trays provided in the ventilation hood at the back of
the room. NEVER pour liquid cultures down the drain.
• Deposit all disposable glassware (broken tubes [sans caps], slides, and other specified material)
in the white "Glass Disposal" receptacle.
• Place all disposable non-glass materials (i.e., plastic transfer and serological pipettes, Petri
Dishes, cotton swabs, etc.) in the white "Plastics Disposal" receptacle.

6. Safety goggles must be worn during the entire lab period. Lab coats are optional

7. If an injury occurs, notify the instructor immediately. Pre-existing cuts scratches or burns should
be covered with a bandage before any lab work is performed.

8. Work only at your designated lab table. You will share laboratory supplies with other sections of
this class. Missing or broken materials should be replaced promptly (see your instructor). You are
responsible for using, cleaning and storing your microscope properly.

8. Only students registered in Bio 240 (no visitors!) will be permitted in this classroom/lab.
These procedures, as well as common sense, must be constantly employed to prevent personal
contamination and/or injury.

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Please record special procedures here as directed by your instructor.

Notes:

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 Exercise 1

The Compound Light Microscope

Introduction.

The compound light microscope is one of the most important tools of the microbiology laboratory. The
microscopes used in this laboratory consist of lenses that magnify images up to 1000X their actual size and can
resolve images as small as 250 nm (0. m). Mechanical adjustments and supportive features of the microscope
afford a broad range of possibilities for viewing the various types of microorganisms.

The microscope combines the principles of an optical system and an illumination system to achieve
magnification in a bright field. Light, projected toward an object, bounces off the object and passes through a
lens called the objective lens to form a magnified image. The image becomes an object for a second lens, the
ocular lens, which magnifies it further and forms an image visible to the observer. This image appears to be as
distant from the eye as a page being read by a person with normal vision. This is a virtual image since it cannot
be projected onto a screen.

Essential aspects of microscopy that ensure proper imaging include:

1) Resolving power or resolution—the ability to distinguish objects that are close together--determines
the size of the smallest object that can be discerned under operating conditions.
2) Working distance refers to the distance between the slide and the bottom of the objective lens.
3) Diameter of field is the width of the diameter of the circle of light that you see when you look through
the ocular lens. As magnification increases, the diameter of field decreases.
4) Refraction pertains to the degree to which light bends as it passes from one medium (glass, oil, or air)
to another during formation and projection of an image through a microscopic lens.

In this exercise the basic features of the microscope will be explored as you gain experience in the use of this
instrument. Prepared slides of various microorganisms will be viewed from which you will prepare sketches.

Materials

• Compound light microscope

• Prepared slides of various microorganisms

Procedure

1. Follow your professor’s instructions regarding the proper methods for transporting, using, and storing your
microscope. Move the microscope and its power cord from its current position on the table and place it on
the desk as instructed. Remove the protective plastic cover, fold neatly and place it in the “cubby”
immediately below your workstation surface. You should clean your scope’s lenses with lens paper and lens
cleaner before and after each lab session.

2. Note the position of the ocular lens (the eyepiece), the various objectives, the stage and slide holder, the
condenser and iris diaphragm, the course and fine adjustments and the light source in the illustration.
Complete the function description of each component in the space provided in the results section.

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3. The total magnification of the different lens systems is calculated as the product of the ocular and objective
magnifications. The magnification possible with each objective should be determined.

4. A variety of prepared slides of microorganisms are available for viewing. Select one to use while
familiarizing yourself with the microscope and its operation. Begin observations with the low power
objective. (HINT: Locate a prominent object in your field of view and use the “slide caddy” to position this
object in the visual center of your field of view. When you move to the next higher magnification, the object
will be at or near the center of your field of view.) Adjust the iris diaphragm to achieve optional lighting.
Keep both eyes open when viewing the specimen through your ocular lens.

5. Place the prepared slide on the stage and locate the area of color with the low power (10x) objective. Tiny
specks will be visible. You can be certain that the proper object is being viewed if the image moves as the
slide is moved. This is important because imperfections on the lens or other debris on the microscope may
be mistaken for the object. The slide also may be viewed at high power.

6. Your microscope is parfocal, which means an object in view under a low power objective should
still be in view under a higher power lens. Therefore, only minor adjustments to the fine focus knob
are needed when you move from a low to a higher power objective., As you increase magnification
power, the diameter of the field of view will decrease. In other words, the diameter of the field of
view is inversely proportional to the magnification. Also, more light will be required to view the
specimen (YOU CAN MOVE THE IRIS DIAPHRAGM CONTROL LEVER, LOCATED UNDER THE
STAGE, TO THE LEFT TO ALLOW MORE LIGHT TO REACH YOUR EYE – DO NOT INCREASE THE
INTENSITY OF THE LIGHT ITSELF, IT SHOULD REMAIN AT 3.5 AT ALL TIMES).

7. After locating and focusing the object of interest using the low power (10X) objective, you will be ready
to use the oil immersion (100X) objective. To achieve the high level of magnification it affords, immersion
oil (a special type of synthetic oil) is placed on the slide between the specimen and the oil immersion
(100X) objective. This oil has the same refractive index as glass. The effect is to prevent light rays from
bending as they pass through the slide to the objective lens. More light is collected by the objective
lens, increasing the numerical aperture of the lens and improving the resolving power of the
microscope. The resolving power is the amount of detail that can be ascertained by the observer and
also refers to minimum distance between two distinct points that the observer can distinguish.

9. With the image centered and focused under the low power (10X) objective, rotate the 10x objective away and
place the scanning power (4X) objective into place (DO NOT ADJUST THE FOCUS KNOBS-REMEMBER
PARFOCAL!). Apply a drop of immersion oil to the viewing area. Open the iris diaphragm fully (slide the
lever under the stage to the left) and swing the oil immersion oil (100X) objective into position. A minor
adjustment with the fine adjustment knob should bring the object into view. It is imperative that the coarse
adjustment not be used with this objective because the slide may crack and the objective may be
damaged. Note that the working distance (the distance between the front of the microscope objective
lens and the surface of the specimen when it is in focus) decreases as total magnification increases due to
the higher numerical apertures associated with high power objectives. If the object cannot be found after
a few moments, leave the oil in place, return to the low power objective, relocate the object, and then move
back to the oil immersion objective. Be careful not to place the high power (40X) objective in the oil- always
move from 10X to 4X to 100X, avoiding the 40X objective.

9. While viewing under oil immersion (1000X total magnification) make sketches of the microorganisms in the
spaces provided below. Determine the size of individual cells using the ocular micrometer. Remember that
the distance of one ocular space when using the oil immersion lens is 1 m. Record in the spaces provided.

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 10. When your work with the microscope is completed, remove the oil from the oil immersion lens by wiping
with lens paper to which you have added two drops of lens cleaning solution. The other lenses should be
cleaned as well (clean the ocular lenses with 70% ethanol and the objective lenses with lens cleaning solution).
Turn the power switch off. Wrap the power cord around the spindle located on the back of the microscope.
Position the scanning (4X) objective in the viewing position, center the stage, and lower it away from the
objectives array in the nosepiece. Position the iris diaphragm lever to the right (0.1) and open the field iris
diaphragm fully (turn to the left). Replace the microscope cover. Return the scope to its original position on
your lab table by lifting it, not dragging it. Replace the microscope cover.

Care of the Olympus

CX-31 Microscope

Using The CX-31:

1. Familiarize yourself with the components of the Olympus scope (see figure on next page).

2. Carefully lift the scope off the table with both hands. The scope should always be covered, as it is when you
approach your workstation. Note the location and storage of the cord.

3. Unwrap the power cord and plug it into the outlet located in the center of your lab table. Turn the power switch
to the on position. Adjust the voltage control as necessary, but never higher than the “light bulb” symbol on the
dial above the on/off switch located to the right of the stage.

4. Adjust the inter-pupillary distance as demonstrated by your professor

5. Familiarize yourself with the operation of the mechanical stage. Note the operation of the specimen holder.

6. Practice operating the coarse and fine adjustment dials by rotating them in a clockwise direction.

7. Practice positioning the iris diaphragm and condenser. Begin with the iris diaphragm in the rightmost position
(at low power) and move it to the left as you proceed to higher power objectives. In most cases, the condenser
should be positioned at its highest point, without touching the underside of your slide, just beneath the stage.

8. Refer to the procedure on page 9 of this manual to be sure you are following all of the steps to use the microscope
successfully.

Storing the CX-31:

1. Turn the power switch off. Wipe all objective lenses and the stage with the lens tissue and lens cleaning solution.
Wipe the ocular lenses with lens tissue and 70% ethanol.

2. Position the Scanning (4X) objective in the viewing position and lower the stage fully away from the objectives
array in the nosepiece.

3. Position the iris diaphragm lever to the right (0.1) and open the field iris diaphragm fully (turn to the left).

4. Wind the power cord securely around the spindle located on the back of the scope. Return the scope to its original
position on your lab table. Replace the microscope cover.

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 Microscope Use Procedure
Follow these steps whenever you use your microscope:

OBSERVATION OF A SPECIMEN
1. Reposition your microscope away from the center of the lab bench by
lifting it, not dragging it, toward you. Uncover it. Place the cover into the
cubby under the lab bench for storage.

2. Clean the ocular and objective lenses with LENS PAPER.

3. Insert the power cord into the outlet and turn on the light, adjust
intensity to 3.5.

4. Open the slide holder and insert the slide into position.
5. Swing in the 10X objective into position and focus using the course and
fine adjustment knobs.

6. Adjust inter-pupillary distance as necessary.

7. Focus first using your right eye only, then adjust the diopter until the
image is in focus using your left eye.
8. Adjust aperture of the iris diaphragm. The lever should be positioned
toward the right when using the 10X objective.
9. Swing the 4X objective into place and place a small drop of immersion oil
on the specimen – do not adjust the focus.
10. Place a small drop of immersion oil on the specimen and swing the oil
immersion (100X) lens into position. Focus using fine adjustment only. Move
the iris diaphragm lever to the left to increase the amount of light.

MICROSCOPE CLEANUP, STORAGE

1. Remove the slide, clean it with lens paper and glass cleaner for prepared
slides (or powdered borax and water for your smears) and put it away.
2. Clean all objective lenses and the stage with lens paper ONLY and lens
cleaning fluid.

3. Clean ocular lenses with lens paper ONLY and 70% ethanol.
3. Turn the light off, coil the power cord around the spindle on the back.
The light intensity knob should remain at 3.5.
4. Position the scanning (4X) lens in the viewing position, center the stage
and lower it away from the objective array of the nose piece.

5. Turn the iris diaphragm lever all the way to the right (0.1 setting).
6. Carefully place your microscope in its original location on the lab table.
DO NOT TO DRAG YOUR SCOPE ACROSS THE TABLE. Lift it off the
table and place it gently in its original position and replace the cover.

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Exercise 1 Questions Name__________________
Section #_______________

1. Explain the function of oil in oil immersion microscopy.

2. Trace the pathway of light from the light source to the eye (describe) and show how the image is formed
by the microscope (draw)

3. What routine procedures should be performed each lab period to care for the microscope properly?

4. The microscopes used in this class are parfocal. How is that important to you as you use your
microscope?

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Exercise 1 Questions Name__________________
Section #_______________

5. Define the following terms:

resolving power –

working distance –

diameter of field –

6. Complete the Following:

Microscope Component Function/Location

Ocular

Objective

Mechanical Stage

Condenser

Iris Diaphragm

Course/Fine Adjustment

Ocular Micrometer

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Exercise 1 Questions Name__________________
Section #_______________

7.

Specimen: Specimen:

Size (um): Size (um):

Specimen: Specimen:

Size (um): Size (um):

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 Exercise 2
Colonial and Cellular Morphologies of Microorganisms &
Occurrence of Microbes in Our Environment

Introduction:

Microscopic life can be found virtually everywhere. Bacteria, fungi, and other forms can be isolated
from the air we breathe, the water we drink, the food we eat, and everything with which we have
contact - including one another.

In this lab, you will sample aspects of your physical environment and your own person to demonstrate the
widespread presence of microorganisms in our world.

Materials:

One 100 mm nutrient agar (NA) plate per student

Sterile cotton swabs
Sterile water

Procedure Day 1:

1. Divide your plate into two halves. Label one half with an “E” for an environmental sample, e.g.,
washbasin, water fountain, etc. Label the second half “P” for your personal sample, e.g., between your
fingers or toes, belly button or earring, etc.

2. Remove one sterile cotton swab from the packet. Dip it in sterile water before collecting your sample (if
collecting from a dry surface). Be sure to remove excess water from swab by pressing it against the side
of the test tube as you remove it.

3. Capture sample by rolling dampened swab over selected area.

4. Tip the lid of your NA plate and roll the swab across the agar surface. Be careful not to tear or puncture
the surface of the agar.

5. Repeat steps 2-4 for your second sample. Close plate and incubate lid down at 37C for 48 hours.

6. Discard contaminated swab in waste container labeled "Plastics".

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Procedure Day 2:

During the 48-hour incubation period any cells deposited when you inoculated each plate will have increased
in number to form a population of cells called a colony. Colonies often present unique characteristics by which
they can be distinguished from one another.

These characteristics include size and shape of the colony, elevation, form, margin, color, appearance (dry, shiny,
wrinkled), See the figures below for examples of some of these morphological characteristics.

1. Remove plates from incubator.

2. Examine each plate for the presence of colonies of microorganisms.

• Describe three or more colonies that are visibly distinct from one another using the terms for form,
elevation, and margin in the figure below. Record your results in the data table on the next page.
Procedure Day 2:
Retrieve your plate from the incubator and examine it for the appearance of microbial growth. What
you see on your plates are called colonies—masses of cells derived from single cells transferred to the
agar medium from your fingertips. An individual colony consists of millions of cells. Different types
of bacteria produce colonies that look differently when compared to each other. The overall
appearance of a microbial colony is called colonial morphology. Use the list below to help you to
describe the colonial morphologies you see on your plate:

Describing Colony Morphology (from David R. Caprette, Teaching Professor, Rice University,
http://www.ruf.rice.edu/~bioslabs/BIOC318/morphology.asp)

As we document our discoveries it is absolutely essential that we use common adjectives to describe
the physical characteristics of the colonies that our isolates form when cultivated on an agar surface.
Below is an illustrated guide to the terms we will use.

1. Culture conditions. Because colony morphology may be influenced by the conditions under which
the species is cultured, it is important to describe those conditions accurately. Include the medium,
temperature of incubation, age of the culture.

2. Form. Irregular colonies that spread rapidly over a plate should be identified as spreading in
addition to irregular. A branching structure distinguishes rhizoid from filamentous. Punctiform
colonies are distinguished from circular colonies by their very small size. Spindle colonies are lens-
shaped.

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3. Elevation. There are no clear distinctions among raised, convex, and pulvinate. Convex would be
close to semi-circular in cross section, while raised and pulvinate colonies have shallower and deeper
profiles, respectively. The "bump" on an umbonate colony is called an umbo.

4. Margin. Erose is synonymous with serrated.

5. Surface. Surface can be smooth, glistening, rough, wrinkled, dry, powdery, moist, mucoid
(forming large moist sticky colonies, brittle, viscous (difficult to remove from loop), butyrous
(buttery).

6. Opacity. Colonies may be transparent (clear), translucent (can see through them), or opaque
(colony blocks transmitted light).

7. Color. White, buff, brown, red, yellow, pink, purple, etc.

8. Size. The size of the colony can be a useful characteristic for identification. The diameter of a
representative colony may be measured in millimeters or described in relative terms such as
pinpoint, small, medium, large.

9. Other. Describe any distinctive odor, any diffusible pigments (staining the agar), or any other
likely distinguishing characteristic.

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Exercise 2 Questions Name__________________
Section #_______________

Select a few colonies for describing. Fill in the following table with descriptions of the bacterial colonies. Use
a separate line for a variety of colonies. HINT: Use color as your primary discriminator, then sort according
to other characteristics. See example below.

EXAMPLE:

Site Color Diameter Form Elevation Margin Appearance

(i.e., floor, (mm)
finger)

Toe jam white < 2 mm Circular Convex Entire Glossy, smooth

yellow 3-8 mm Irregular Flat Undulate Grainy, dry

Colony Description
Site Color Diameter Form Elevation Margin Appearance
(i.e., floor, (mm)
finger)

Questions:
2. Approximately how many different types of bacteria appear to be present on each of your plates?
How do you know?

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Exercise 2 Questions Name__________________
Section #_______________

3. Do you think all the organisms living at your sample sites grew on your nutrient agar? Briefly discuss.

________________________________________________________________________________________________

___________________________________________________________________________________________________

___________________________________________________________________________________________________

__________________________________________

4. Of what advantage is a solid medium such as nutrient agar and Petri plates?
_______________________________________________________________________________

__________________________________________________________________________________________________

__________________________________________________________________________________________________

_____________________________________________________

5. Of what advantage is a liquid medium such as nutrient broth?

___________________________________________________________________________________________________
__________________________________________________________________________________________________
_____________________________________________________

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 Exercise 3

Effectiveness of Hand Washing

Introduction.

Vast numbers of microbial hitchhikers populate our bodies. These microscopic passengers are
comprised to two major groups—permanent residents that make up our normal flora-- and temporary
visitors that may be present for only a few days or weeks. This latter group is described as our transient
flora. Simple washing can remove some of the transient flora present on our hands, depending on how
the wash is done.

Members of each group can be found on and within our bodies at any time. These organisms, of course,
can be transferred from one person to another by various means. In 1846, Ignatz Semmelweis noted a
strong correlation between puerperal (childbed) fever and the fact that caregivers went from patient to
patient or from autopsy to patient without first washing their hands. After implementing a strict
protocol for caregivers to disinfect their hands between patient examinations, the incidence of childbed
fever and other infections declined dramatically.

Materials.

1 nutrient agar plate per student

various antimicrobial hand soaps

Procedure Day 1:

1. Use a wax marker to divide each NA plate into two equal halves. Be sure to mark the bottom of
the plate only. Label one half “Before” and the other “After.” Be sure to put your initials and class
time on the bottom of the plate as well.

Before After

Initials Section #

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2. Prior to hand washing tip the lid of your plate (i.e., do not open the lid completely as this may allow
dust or microbes to enter!) and touch the “Before” section with your fingertips.

3. Two of the individuals on each table will wash their hands, the other two will use hand sanitizer
only. Please make sure you record which one you are doing.
Hand washing instructions: Wash your hands well with one of the soaps provided and water. Rinse,
shake off the excess water (do not dry your hands with paper towel) and touch the “After” section
with the same hand used to inoculate the “Before” section.
Hand sanitizer instructions: squirt hand sanitizer onto your hands and rub thoroughly all over the
hands. Touch “After” section with the same hand used to inoculate the Before section.

4. Place your inverted plate in the designated incubator until the next class meeting.

Procedure Day 2:
Retrieve your plate from the incubator and examine it for the appearance of microbial growth.

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Exercise 3 Questions - Handwashing. Name ________________
Section # _____________

Sketch the results from the "Hand washing" activity.

Soap 1 (name) _____________
Soap 2 (name) _____________
Hand sanitizer 1 (name) _____________;
% ethanol ___________
Before After Hand sanitizer 2 (name) _____________;
% ethanol ___________

1. Describe 4 distinct colonies in the table below:

Colony 1 Colony 2 Colony 3 Colony 4

Source (before
or after) (soap
or hand
sanitizer)
Culture
Conditions
Form

Elevation

Margin

Surface

Opacity

Color

Size (in mm)

Other traits

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2. Compare growth results on the “BEFORE “and “AFTER” sections.
Which plate appears to have more microbial growth?
Which plate appears to have more different types of microbes?
How many different types of microbes on each plate?

3. Within your group compare the handwashing results with those of the hand sanitizer.
Which plates had the greatest reduction in number of microbes after treatment?
Which plates had the greatest reduction in variety of microbes after treatment?

4. Using what you know about handwashing and hand sanitizer explain your results in a short
paragraph (minimum 6 sentences). You may wish to include information about mode of action of hand
sanitizer and hand washing in your explanation of the results.

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 Exercise 4

Simple Staining

Simple Stain Procedure:

Select an individual colony that is well separated from others on the agar surface of your hand washing
plate. You’ll use this colony to prepare a simple stain so that you can observe the organism’s cellular
morphology (appearance of an individual bacterial cell as viewed using the microscope) as well as its
colonial morphology (appearance of a mass of cells growing on nutrient agar). Most bacteria are tiny
(1-10 um), and many are nearly transparent. Obviously, they are difficult to visualize even with a good
light microscope. Staining bacterial cells increases their contrast so that aspects of cellular
morphology—size, shape and arrangement—can be determined. By itself, staining cannot be used to
identify an organism with any certainty, but information about cell morphology and colonial
morphology is helpful when combined with other identification procedures.

Materials:

Cultures from “Hand Washing” lab

Microscope slides, stains

A. Preparation of a bacterial smear.

1. Clean a glass slide with powdered soap located near the sink. Blot dry with Bibulous Paper.
Capture a loop full of de-ionized water from the water bottle located in your Stain Drawer and transfer
it to the center of your slide:

2. Transfer bacteria from a single colony into the water drop on your slide using a loop sterilized in
the Bacti-Cinerator as demonstrated by your instructor. The margin of a colony provides the “best”
cells.
HINT: The thinner the smear, the better your staining results will be.

3. Allow the smear to air dry completely.

4. Heat fix the smear by placing the slide on the platform attached to your incinerator cylinder for 30-
60 seconds—excess heat can distort the cells. Let slide cool for 30-60 seconds.

5. Stain the smear by covering with 3 drops of crystal violet or safranin for (30-60 seconds). Rinse
with distilled water from squeeze bottle into the lab sink. Be sure to hold your slide at an angle and to
rinse above the smear, letting the water flow over your sample. Blot dry with bibulous paper.

6. View the stained slide under oil immersion.

7. Describe the colonial and cellular morphology and diagram a representative arrangement of the
cells from the colony you selected in the space provided for “colony 1” in the section “Exercise 3
Questions – Simple Stain.” Measure the diameter and/or length of the cells examined. It is helpful

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to measure several individual cells and then determine an average size or a size range. Measure at
least 10 cells to make these determinations.

10. Select another colony that presents a different appearance than the first one examined. If no
different colonies appear on your plates, use your lab partner’s plate or the plate of another
student. Prepare a simple stain of this colony. Describe the colonial and cellular morphology and
diagram a representative arrangement of the cells from the colony you selected in the space
provided for “colony 2.”

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Exercise 4 Questions – Simple Stain. Name ____________________
Section # _________________

Cellular observations should be made under oil immersion. Observe and describe the cellular size, shape and arrangement
of your specimen. Helpful hints: The arrangement is the one that seems most prevalent. It is not unusual to see two or
more arrangements on any given slide. On oil immersion a single unit of your ocular micrometer is equal to 1 um.

Diagram a representative arrangement of the cells from the colony you selected in the space provided. Measure the
diameter and/or length of the cells examined. It is helpful to measure several individual cells and then determine an
average size or a size range. Measure at least 10 cells to make these determinations.

Colony 1
Source____________________

Cell size__________________

Cell Shape ________________

Cell arrangement ______________

Colony description
________________________________________
________________________________________
________________________________________

Colony 1 – sketch of cells

Colony 2
Source____________________

Cell size__________________

Cell Shape ________________

Cell arrangement ______________

Colony description
______________________________________
______________________________________
______________________________________

Colony 2 – sketch of cells

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Exercise 4 Questions – Simple Stain. Name ____________________
Section # _________________

1. Distinguish between colonial morphology and cellular morphology.

Do you expect that different organisms will vary in these characteristics?
How can observations about these morphologies facilitate identification of a specific organism?

2. Heat fixing the smear before staining has three functions. Name all of them.

3. Describe the properties of the stain and the bacterial cell which cause the stain to adhere to the cells.

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 Exercise 5
Differential Staining
Introduction.

Identification of bacteria typically begins with one or more differential stains. These stains
differentiate bacterial cells on a morphological or structural level. One such stain—the Gram Stain
will be explored in this lab.

The Gram Stain:

The Gram stain is one of the most significant techniques used to identify members of this diverse
kingdom recognized as the Eubacteria.

Hans Christian Gram discovered the Gram staining technique in 1884. He hoped to develop a
procedure through which pathogenic (disease-causing) bacteria could be distinguished from human
lung tissue. Though the procedure didn’t work as anticipated, Gram noticed that bacteria retained one
of the two reagents used. Those that retain the primary stain (crystal violet) are described as Gram
positive bacteria. Those that retain the secondary or counter stain (safranin) are described as Gram
negative.

The Gram reaction depends in large part on the nature of the bacterial cell wall. Gram positive bacteria
possess a cell wall that contains a thick layer of peptidoglycan. This protein/polysaccharide complex
seems to retain crystal violet more readily than do Gram negative cells that contain a great deal of
phospholipid components and very little peptidoglycan in their cell walls.

Some genera of bacteria can be described as Gram variable or Gram non-reactives. Bacteria in the
genera Bacillus, Micrococcus, Neisseria and Moraxella can display atypical Gram reactions. Members of
the genus Mycobacterium are unpredictable because of the high concentration of mycolic acid in their
cell walls. Mycoplasma species, on the other hand, cannot be Gram stained because they lack cell walls.

Procedure:

The Gram Stain: Use colonies from fresh cultures provided. Check with your instructor to be sure
which cultures are appropriate. Prepare a bacterial smear from an isolated colony as described in the
simple stain lab. Remember: Colonies that look different generally are derived from individual cells that
are different species.

1. Air dry your smear then heat fix it by placing the slide on the metal platform on top of your Bacti-
Cinerator for 30-60 seconds. This will help bind the cells to the slide, kill them, and increase the
uptake of stain by the cells.

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Figure 1: Your Bacti--Cinerator with heat-fixing platform attached to it.

2. Let the slide cool for at least 30 seconds before applying first stain.
3. Cover with 2 to 3 drops of crystal violet (30-60 seconds)
4. Rinse with distilled water.
5. Cover with 2 to 3 drops of iodine (mordant) for 1 minute.
6. Rinse with distilled water.
7. Decolorize with alcohol/acetone for < 5 seconds and rinse with water.
8. Cover with 2 to 3 drops of safranin (1 minute)
9. Rinse, blot dry with bibulous paper and view under oil immersion.

31
 Exercise 5 Questions: Name _______________________
Section # ____________________

1. Complete the following table for your Gram stain procedure:

Gram
Bacterium Size
reaction Shape Arrangement
(Genus species) (µm)
(+ or -)
#1
#2
#3

2. What does iodine do in this procedure?

3. Why do Gram positive cells retain crystal violet while Gram negative cells do not?

4. Does the Gram stain procedure work for all bacteria? Why or why not? Answer must be at least
three sentences.

32
 Exercise 5 Questions: Name _______________________
Section # ____________________

5. Which Gram stain would your body’s cells (eukaryotic cells) most likely retain? Explain?

(minimum two sentences)

6. Why is it important for a doctor to know the results of differential stain tests in terms of deciding
how to treat a patient’s infection? Answer must be a paragraph of at least 6 sentences and must
include some discussion of antibiotics and common infections.

33
34
Lab Practical # 1 Name______________________

Microscopy and Gram Staining

Directions: No. No.

1. Obtain 2 test cultures from the supply area. Be sure to pick one even
numbered culture and one odd numbered one.
Colonial Morphology (5 pts. each)
2. Describe the Colonial Morphology for each of your cultures. Score
No.

No.

Cellular Morphology (10 pts. each)

3. Describe the cellular morphology, arrangement, and size of the organisms in your cultures.
No.

No.

Gram Reaction (5 pts each)

4. Gram reaction (positive or negative).

No.

No.
Proficiency, Safety and Clean-up (10 points)
5. Scoring on general laboratory procedures.
Instructor
Comments:

Totals Score:

35
36
 Exercise 6

Isolation of a Pure Culture by the Streak Plate Method

Introduction:

You have already seen that different bacteria grow in macroscopic groupings called colonies. Like
individual cells, these colonies display their own unique morphologies.

Aspects of colonial morphology include elevation, margin, form, optical characteristics (opaque,
translucent or transparent), texture, pigmentation and size. Because each organism tends to produce a
characteristic morphology, these aspects can be used to distinguish those organisms found in a mixed
culture (culture containing more than one species). By successfully isolating such colonies you can
establish pure cultures (culture containing only one species) for subsequent identification and testing.

One technique used to achieve such isolation is called the streak plate method. In today’s lab, you’ll
use Nutrient Agar (NA) plates to isolate organisms from broth cultures that contain two bacteria. Once
you have achieved isolation, you’ll also have the opportunity to further develop your Gram staining
skills and your observations of cellular morphologies.

Materials:

One nutrient agar plate per student

One (numbered) mixed broth culture per every 2 students containing two of the following
bacteria:

Staphylococcus epidermidis (forms circular, white colonies)

Serratia marcescens (forms irregular red colonies)

Always be careful not to spill the broth!

Procedures:

Day 1:

1. Label the bottom of your plate with your initials, the culture number and date.
2. Also on the bottom of your plate, separate it into three sections as shown in the illustrations that
follow.

37
Inoculate the isolation plate using the following procedures (see illustration above):

3. Sterilize your inoculating loop in the Bacti-Cinerator. Heat the wire until it glows red. Do not
lay down the loop once it has been sterilized.
4. While the loop is cooling for a few seconds, pick up your mixed culture broth. Mix the broth with
the Vortex mixer to re-suspend the cells if necessary.
5. Dip your sterilized loop into the broth to “capture” your cells.
6. Lift the lid of your plate and gently streak on the surface of the agar in section 1. Be sure to use all
the surface area of section 1 to ensure that cells are physically separated. Remove your loop and cover
the plate.
7. Sterilize your loop as described in (1.). Do not dip loop into broth again.
8. Streak across section 1 once, dragging cells into section 2.
9. Sterilize loop. Repeat previous step taking cells from section 2 to section 3.
10. Invert the plate before placing it in the incubator until next lab period. This plate is termed an
isolation plate.

Note: your instructor may prefer that you divide your isolation plate into four sections as shown below:

38
Day 2:

1. Examine your plate to observe colonies that differ from one another. Compare isolated colonies on
your plate with pure cultures of each organism provided. Compile relevant observations in the space
provided.
2. Select two isolated, distinctly different colonies from which to prepare smears for Gram staining.

39
 Exercise 6 Questions: Name _______________________
Section # ____________________

Complete the following table:

Name Colonial Morphology Cellular Morphology and Gram Rx

arrangement.

1. How would you know if you had a contaminant colony on your plate?

2. Name and briefly describe three common technique errors that might lead to a failure of the streak
plate method to produce a pure culture?

3. Why is the streak plate so important in the production of pure cultures?

40
41
 Exercise 7

Use of the API 20E System to Identify Gram Negative Bacteria

Introduction:

The API System is a convenient battery of tests for identification of members of the family of bacteria known as
Enterobacteriaceae. Some of the genera of this family are intestinal pathogens. Included in the family are
Enterobacter, Shigella, Salmonella, Escherichia, Klebsiella, Proteus, and Yersinia. Up to 23 tests can be conducted by
inoculating 20 “tubes” in a plastic strip as shown here:

Figure 1. The API Test Strip

The test culture is grown as a pure culture on nutrient agar for 24-48 hours. Follow the instructions below for
identification. Record your results in the figure or on separate sheets provided in the lab.

Materials:

sterile transfer pipette

sterile cotton swab
pure culture of test organism
one ampule of 0.85% sterile saline per every 4 students (i.e., per table; 6/section, maximum)
API 20E test strip, tray, and cover per every 4 students (i.e., per table; 6/section, maximum)
tap water
sterile mineral oil
test reagents

42
Procedure - First Day - “Inoculation of the Strip” (note that this will be performed for you; you will begin
the lab with the second day procedure “Reading the Strip”):

1. Prepare a saline suspension of your test organism by transferring a single colony from the agar dish to a sterile
saline ampule with your sterile swab. Mix the suspension well.

2. Label the end of the API strip with your lab group # and the name (or number) of the test organism.

3. Dispense about 5ml of water into the base of the tray. This provides humidity so the test strip does not
dehydrate.
4. Remove an API test strip from the sealed pouch (be sure to reseal the pouch) and place your test strip in your
tray. Water level should not be above the strip.

5. You will now transfer sample from the saline ampule to the API “tubes”. Follow directions precisely. Tilt
the API tray and carefully load the tubules as follows:

Follow directions precisely. Fill the underlined tubes (ADH, LDC, ODC, H2S, and URE) up to the line that
divides the cupule from the tubule. [See Figure 2] Cover these tubes with sterile mineral oil by placing 2-3 drops
of oil in the cupule as demonstrated by your instructor. The oil covering provides anaerobic conditions.

cupule
Figure 2:

Fill to this level

tube

Follow directions precisely. Completely fill the cupule and tubes of the |CIT| , |VP| , and |GEL| chambers
with saline suspension of your culture. These tubes are indicated with a "goalpost" in Figure 1. This allows
growth in aerobic conditions.

Follow directions precisely. All other chambers are to be filled to the top of the tube - not into the cupule.

6. Place the lid on the incubation tray and incubate at 37° C for 18-24 hrs.

43
Procedure – Second Day - “Reading the Strip”:

Materials for Second Day.

incubation try inoculated on day one 10% ferric chloride (TDA)

Barritt’s reagent A &B (VP 1 & VP 2) Kovac’s reagent (IND)
sterile transfer pipettes

1. Refer to Chart (handout - see your instructor) on the following page. Determine results (as positive or negative)
for all reactions not requiring reagents. Record
(+) or (-) on the test sheet provided.

2. Add 1 drop of TDA reagent (10% ferric chloride) to the TDA tube. A positive reaction (dark brown-red) will
occur immediately. A negative reaction is yellow.

3. Add 1 drop each of VP1 and VP2 reagents (Barritt’s A and Barritt's B) to the VP tube. A positive reaction (red)
may take 10 minutes to appear. If it turns light pink within a couple of minutes and continues to darken, record
the test result at positive. If the solution remains colorless, or turns pink after 10 minutes, interpret it as negative.

4. Add 1 drop of IND reagent (Kovac’s reagent) to the IND tube. A red ring will form within about 2 minutes if
the test is positive. After several more minutes the tube may turn from yellow to brown, which is negative.

5. Examine the GLU tube. If it changed to yellow or gray record the GLU test as positive.

6. Please note that the tubes are grouped in sets of three in the figure above. For each positive result record the
value give 0 and 7. Now determine the 7-digit profile number for your test organism. The last digit (NO2 and
N2) is not used in our sample.

7. Determine the identity of your test organism by looking up the 7-digit profile number on the API website on
the classroom computer.

Identification: Likelihood:

8. When finished place the test strip in the biohazard container at the back of the lab. Place all reagents in the
proper place on the preparation table. Note: There are no questions to be completed for this exercise.

44
45
 Lab Practical # 2
Identification of the Laboratory Unknown

DAY 1

1. Each unknown consists of 2 species, one G+ and one G- mixed in a nutrient broth culture. Students will work
in pairs or you may work alone if you wish. Please ask your instructor for permission to have more than two
students in a group. Select an unknown from the broth cultures provided. Confer with your astrologer or
psychic - some are more difficult than others!

2. Begin isolation procedures. Use the streak plate method. Each partner should make one plate. This is the
crucial step.

3. Transfer about 4ml of your culture to a cuvette and determine (spectrophotometrically) the CFU/mL of your
culture. Discard the contents of the cuvette in the disinfectant container provided.

4. Be sure all plates are labeled properly with your initials and culture number. Incubate until the next lab
period.

DAY 2

1. Gram stain the backup culture (slant) produced on day one. Confirm the presence of G+ and G- cells. Store in
the refrigerator as a backup culture.

2. Observe your streak plates for well-isolated colonies of distinct morphologies. Prepare Gram stains of these
colonies to confirm they are different (e.g., G+ or G-).

3. Streak (for growth) each of your isolates to a fresh NA plate. Place both streaks on a single plate and label as
shown. This plate will be the source from which you will transfer for differential testing. Also, you will turn
this plate in for scoring.

G+ G-

Name Date
Culture #

4. Incubate your isolation plate at 37°C until the next lab period.

DAY 3

1. Observe your isolation plate for colonies of distinct morphologies. Gram stain these colonies to confirm that
they are different (e.g., G+ or G-).
46
2. Based on the results of your Gram stain transfer the Gram negative to an API test strip for identification.
Incubate your API test strip for 24 hours.

3. Differentiate the Gram-positive organism by performing the Accu-Staph test. Record results on the Lab
Practical #3 report sheet.

47
Lab Practical # 2 Name(s)__________&__________Culture #____

Colonial Morphology (5 pts. each)

1. Describe the Colonial Morphology for each of your cultures. Score

No.

No.

Cellular Morphology and Arrangement (10pts. each)

3. Describe the cellular morphology, size and arrangement the organisms in your culture.

No.

No.

API Formula and Accu-Staph Results. (5 pts. each)

4. Record the API Formula and Sketch the Accu-Staph Results Below.

No.

No.

Identification of the Organism (5 pts each)

5. Name the Organisms Below.

No.

No.

Instructor Comments and Final Score:

48
49
 Exercise 8
Wedding Reception Disaster
Materials:
Day 1: Phenol Red Glucose Broth Test

MEDIA PER SECTION:

12 8mL Nutrient Broth Cultures Labeled as Unknowns (A through L)

12 60mm TSA Plates
12 12mL Glucose Broth

NOTE TO INSTRUCTOR: Phenol Red Broth tubes should be removed from the incubator within 24 hours to
prevent false positive

Day 2: Mannitol Agar Test and Urea Broth Test

MEDIA PER SECTION:

6 60mm Mannitol Salt Agar Plates

6 5mL Urea Broth

NOTE TO INSTRUCTOR: Mannitol Salt Agar (MSA) plates should be removed from the incubator within 8 –
18 hours to prevent false positive

Procedure:

MATERIAL CHECKLIST for Students

Day 1:
_____ 2 unknown cultures, labeled A, B, C, D, E, F, G, H I, J, K, or L
_____ 2 TSA plates
_____ 2 glucose broth tubes
_____2 sterile swabs (2 packs of 2 swabs)
_____2 transfer pipettes

Day 2:
_____ TSA plates from day 1 _____ 1 or 2 urea broth tubes
_____ glucose broth tubes from day 1 _____ 1 or 2 mannitol agar plates

Day 3:

_____ mannitol agar plates from day 2 _____ urea broth tubes from day 2

50
The Wedding Reception Disaster (adapted from R. Runs and used with the permission of Professor John
Griffis)

(Portions of the following are fictitious. Names of bacteria and the characteristics of each have NOT been
changed to protect the innocent.)

123 attendees of Ben and Jen’s wedding reception became ill with symptoms of food poisoning (nausea, vomiting
& diarrhea. All 123 of these people had fed upon 12 foods at a supper just prior to experiencing symptoms. Any
of these 12 could have been the source of a bacterial species that caused the poisoning. Bacteria was isolated
from the 12 foods and 4 different species of bacteria were identified - Staphylococcus aureus, is a common cause
of food poisoning; the other 3 are relatively harmless. Unfortunately, the laboratory scrambled up the samples.
Now it’s up to a group of independent investigators (that's you) to determine which sample is which, and what
foods were the source of each bacterium. Although only a single test is necessary to determine which bacterium
was S. aureus your goal is thorough investigation - to identify all bacterial samples so that the source of origin of
each might be determined.

Thankfully the task was made easier in that only 1 bacterial species was found associated with each food. The
labeled samples and the foods from which the samples were derived are as follows:

A – shrimp gazpacho
B – crab cakes
C - watermelon
D – lobster mac n cheese
E - grits
F – mashed potatoes
G – duck confit
H - brisket
I - mixed greens (arugula, kale, spinach)
J - crème brulee
K- peach cobbler
L- heirloom tomato salad

The characteristics of each bacterium species is:

Staphylococcus aureus - found on the skin or on the lining of the nose or throat; about 20% of all people carry
this bacterium; part of normal flora, but also may cause food poisoning, wound infections, pimples, boils,
pneumonia, meningitis and toxic shock syndrome. Resistant to penicillin but treatable with erythromycin,
or other antibiotics. Gram positive; non-motile; spherical, usually 2 attached but may be singly, or in clusters;
colony growth circular, smooth, pigmented. Can ferment glucose; can metabolize mannitol.

Staphylococcus epidermidis - common on skin; rarely pathogenic but can cause skin, wound or blood infections.
Gram positive; non-motile; spherical, occurring singly, paired or in cluster; can ferment glucose; cannot
metabolize mannitol; colonies are smooth, circular and non-pigmented.

Micrococcus is a related bacterium to Staphylococcus. The bacteria is found in soil but can be air borne. It is
non-pathogenic, although it has been found to cause illness in lab animals which have previously been
"germ-free". Gram positive, non-motile, spherical cells often in groups of 4; cannot ferment glucose.

Micrococcus luteus forms yellow colonies; it can metabolize urea. Micrococcus roseus forms pink colonies; it
cannot metabolize urea.

51
NOTES:

Glucose is used for energy production.

Mannitol is an alcohol derivative of the sugar mannose, used as a source of energy by some bacteria.

Phenol red is a pH indicator. It is peach at neutral; pink at basic; and yellow at acidic pH.

Urea is a nitrogenous compound. Some microorganisms have an enzyme for converting urea to ammonia
and carbon dioxide.

Trypticase soy agar (TSA) contains enzymatic digests of milk protein (casein) and soybean meal with agar
added to provide a solid consistency.

PROCEDURE:

1. Wash hands thoroughly with an antibacterial soap. Wear goggles. Also wash the table surfaces thoroughly
with disinfectant. (Note: It is suggested that you remove all rings or other jewelry from your hands and
arms.)

2. Obtain 2 samples of unknown bacteria. Record the letters of your unknowns in the Table on the Flow Chart
page.

3. Make all transfers from unknown samples onto agar plates using a sterile swab or sterile inoculating loop
and into liquid broth tubes using a sterile transfer pipette or sterile inoculating loop, as directed. Be sure to
vortex the unknowns just prior to each transfer. Whenever you transfer bacteria to a culture plate or slant,
the tops should only be removed a minimal amount of time.

1st Day -- Phenol Red Glucose Broth Test.

1. Obtain 2 TSA plates and 2 glucose broth tubes.

2. Label the bottom of one plate with the letter of your first lettered culture, your class section number, and
your group’s initials. Carefully vortex the first unknown culture. Remove the lid and insert a sterile swab
into the culture. Gently press the swab against the interior of the test tube to remove excess liquid.
Remove the swab and replace the lid. Lift the top of the labeled TSA plate and swab the agar in a zig-
zag pattern (Diagram 1). Immediately replace the top on your agar plate. Repeat this procedure for your
second unknown on the other agar plate. Discard used swabs in the biohazard container as designated
by your instructor.

Diagram 1

52
 3. Next, transfer 1 ml of each unknown into each of the two glucose broth tubes using sterile transfer
pipettes. Label a glucose broth tube with the letter of your first unknown. Carefully vortex the first
unknown culture. Remove the lid and withdraw 1 ml using a sterile transfer pipette. Transfer the bacteria
into the glucose broth tube and swirl. Once again, replace the caps as quickly as possible. Repeat this
procedure for your second unknown using a new pipette and the other glucose broth tube. Discard used
pipettes in the biohazard container. Place agar plates and glucose broth tubes in the incubator. As
directed by your instructor.

4. Wash your hands and table when you are finished. Your instructor will direct you where to place
your unknown cultures.

NOTICE TO INSTRUCTORS: Phenol Red Broth tubes should be removed from the incubator within 24
hours to prevent false positives.

Day 2

Phenol Red Glucose Broth Results:

1. Wash hands & table as before.

2. Observe the growth of bacterial colonies on your TSA plates. Record the color & appearance in your data
table. (If no growth, leave "appearance" blank in your data table; your instructor will supply you with a
source of your unknown.)

3. Observe your glucose broth tubes. If the color remains red, glucose was not fermented and your unknown
is one of the two Micrococcus species. If the color has changed to yellow, glucose was fermented and your
unknown is one of the two Staphylococcus species.

4. Dispose of your glucose nutrient broth tubes in the appropriate tray.

If your unknown is Staphylococcus, then set up the Mannitol Agar Test. If your unknown is Micrococcus, set up
the Urea Broth test.

Mannitol Salt Agar (MSA) Test:

1. Obtain 1 (or 2 if necessary) red Mannitol Agar plates.

2. Sterilize your inoculating loop in the Bacti-Cinerator. Open the lid of your TSA plate and obtain a small
amount of bacteria from the edge of growth on your loop. Replace the top of your TSA plate. Transfer
the bacteria to a mannitol agar plate by smearing in a zig-zag pattern as before. Re-sterilize your loop in
the Bacti-Cinerator. Be sure to label the bottom of the mannitol agar plate with the letter of your
unknown, your group’s initials, and your section number. Place plate lid down in the incubator. Repeat
this procedure if necessary for your second unknown on another mannitol agar plate.
-
NOTICE TO INSTRUCTORS: Mannitol Salt Agar (MSA) plates should be removed from the incubator
within 8-18 hours to prevent false positives.

53
Urea Broth Test: This test uses phenol red as a pH indicator. The broth is peach colored at neutral pH but turns
bright pink if urea is utilized.

1. Obtain 1 (or 2, if necessary) urea broth tubes.

2. Sterilize your inoculating loop in the Bacti-Cinerator. Open the lid of your TSA plate and obtain a small
amount of bacteria from the edge of growth on your loop. Replace the top of your TSA plate. Re-sterilize
your loop in the Bacti-Cinerator. Be sure to label the glass of the tube with the letter of your unknown,
your group’s initials, and your section number. Place tube in the incubator. Repeat, if necessary, for your
second unknown.

54
Day 3 Check Results:

MSA Test results: If mannitol was used for energy, the plate turns yellow - this is Staphylococcus aureus. If
mannitol was not used, the plate stays mostly red (compare to other plates that have turned very yellow) and
your unknown is Staphylococcus epidermidis.

Urea Broth Test: If your broth turns pink, your unknown is Micrococcus luteus; if the broth stays peach, your
unknown is Micrococcus roseus.

Record all final results in your data tables. Discard all plates and tubes in the Biohazard bag, and tubes in the
appropriate tray.

FLOW CHART:
Phenol Red Glucose Test

+ -
red to yellow stays red

Staphylococcus Micrococcus

MSA Test Urea Broth Test

+ - + -
red to yellow stays red peach to pink stays peach

S. aureus S. epidermidis M. luteus M. roseus

55
Data Table: N/A = not applicable (did not perform test)
APPEARANCE PHENOL CONCLUSION- CONCLUSION- UREA CONCLUSION-
UNKNOWN OF COLONY RED TEST UNKNOWN MSA UNKNOWN BROTH UNKNOWN
(A-L) GROWTH (+ OR -) GENUS TEST SPECIES TEST SPECIES
(+ OR -) (+ OR -)

______ ______

______ ______

56
Exercise 8 Questions: Name__________________________
Section # _________________

1. Using the class data, identify which bacterium was associated with each food, and which foods were the
source of food poisoning.

Bacterium Species Food Poisoning – YES or NO

A-shrimp gazpacho

B- crabcakes

C- watermelon

D-lobster mac n cheese

E- grits
F-mashed potatoes

G-duck confit

H- brisket
I- mixed greens
J- crème brulee
K- peach cobbler
L- heirloom tomato salad

2. Why were biochemical tests used to distinguish the bacteria that are contaminating the foods served at
the convention? (In other words, why would colony and cellular morphology be inadequate?)

3. In your own words, use 2-3 complete sentences to explain the biochemical significance of a color
change from red to yellow in the phenol red test.

57
4. In your own words, use 2-3 complete sentences to explain the biochemical significance of a color
change from red to yellow in the MSA test.

5. In your own words, use 2-3 complete sentences to explain the biochemical significance of a color
change from peach to pink in the urea broth test.

58
59
 Exercise 9
HIV ELISA
HIV ELISA Materials Checklist

MATERIAL PROVIDED PER SECTION:

6 Set-ups in Large Bin Each Containing:

1 400mL Disposable Beaker “Clean Transfer Pipets”

(Non-sterile graduated disposable transfer pipets, 8/group)
1 400mL Disposable Beaker “Used Transfer Pipets”
2 Wash Buffer Bottles
1 Rack of Pipet Tips
2 Sharpie Makers
2 20-200L Micropipettors (Set at 50L)
2 Lab Quick Guides

ALSO PROVIDE PER SECTION:

1 Bin of 2-fold Paper Towels (Place at Lab Stations)

12 12-Well Microplate Strips Placed in Tiny Bin (2 strips/group)

12 Racks of Reagents (2 racks/group) KEEP REFRIGERATED, DO NOT DISCARD, RETURN TO INSTRUCTOR

Extra Non-Sterile Graduated Disposable Transfer Pipets

LABELED REAGENT MICROTUBES (12 Racks total; 2/group)

KEEP REFRIGERATED, DO NOT DISCARD, RETURN TO INSTRUCTOR

Color of Labeled
Microtube
Violet +
Blue −
Green PA
Orange SA
Amber SUB
Yellow A
Yellow B

60
ELISA Antibody-Antigen test: Detection of HIV

Student Manual (From Bio-Rad, Inc.)

Introduction
Immunology is the study of the immune system and how the body protects itself against disease. Over 100 years
ago, biologists found that animals’ internal immune systems respond to invasion by “foreign entities” or
antigens. When an invader enters the body, it provokes an immune response that begins with the production of
proteins called antibodies. Like magic bullets, antibodies seek out and attach themselves to invading entities
(antigens), flagging the invaders for destruction by other cells of the immune system. The antigenic invaders
may be any molecules foreign to the body, including components of infectious agents like bacteria, viruses, and
fungi. Today, antibodies have become vital scientific tools, used in biotechnology research and to diagnose and
treat disease. The number of different antibodies circulating in the blood has been estimated to be between 106
and 1011, so there is usually an antibody ready to deal with any antigen. In fact, antibodies make up to 15% of
your total blood serum protein. Antibodies are very specific; each antibody recognizes only a single antigen.

You are about to perform an ELISA (enzyme-linked immunosorbent assay). The ELISA relies on antibodies to
detect the presence of antigens in liquid samples. Because they are antibody-based, ELISAs are called
immunoassays. ELISAs can detect minute amounts of disease agents in samples such as body fluids (before the
body has had a chance to mount an immune response). Smallpox virus is an example of a disease agent that can
now be detected using an ELISA. If exposure is detected and treated with vaccine within 2–3 days, patients do
not develop smallpox. Other applications for ELISA include testing for West Nile virus, HIV coat protein p24,
SARS virus, anthrax spores, hormones such as hCG in pregnancy tests, illegal steroids in drug tests, bacteria in
food safety tests, and the presence of genetically modified organisms contaminating non-GMO food.

Where Is ELISA Used in the Real World?

With its rapid test results, the ELISA has had a major impact on many aspects of medicine and agriculture. ELISA
is used for such diverse purposes as home pregnancy tests, disease detection in people, animals, and plants,
detecting illegal drug use, testing indoor air quality, and determining if food is labeled accurately. For new and
emerging diseases like severe acute respiratory syndrome (SARS), one of the highest priorities of the US Centers
for Disease Control and Prevention (CDC) and the World Health Organization (WHO) has been to develop an
ELISA that can quickly and easily verify whether patients have been exposed to the virus. Over-the-counter kits
that are based on the same principles as this ELISA activity include home pregnancy and ovulation tests, and
tests for the presence of illegal drugs like marijuana and cocaine.

Some tests give positive or negative results in a matter of minutes. For example, home pregnancy dipstick tests
detect levels of human chorionic gonadotropin (hCG), a hormone that appears in the blood and urine of pregnant
women within days of fertilization. The wick area of the dipstick is coated with anti-hCG antibody labeled with

61
a pink compound (step 1). When the strip is dipped in urine, if hCG is present it will bind to the pink antibody,
and the pink hCG-antibody complex will migrate up the strip via capillary action (step 2). When the pink
complex reaches the first test zone, a narrow strip containing an unlabeled fixed anti-hCG antibody, the complex
will bind and concentrate there, making a pink stripe (step 3). The dipsticks have a built-in control zone
containing an unlabeled secondary antibody that binds unbound pink complex (present in both positive and
negative results) in the second stripe (step 4). Thus, every valid test will give a second pink stripe, but only a
positive pregnancy test will give two pink stripes.

How Are Antibodies Made?

When exposed to antigens, all mammals generate an immune response and produce antibodies, proteins that
recognize and bind tightly to the specific antigens. Each antibody recognizes only a single antigen. Animals such
as goats, rabbits, and mice can be injected with an antigen and, after a period of time, their serum will contain
antibodies that specifically recognize that antigen. If the antigen was a disease-causing agent, the antibodies can
be used to develop diagnostic tests for the disease. In an immunoassay, the antibodies used to recognize antigens
like disease agents are called primary antibodies. Secondary antibodies recognize and bind to primary
antibodies in an immunoassay. They are prepared by injecting antibodies produced by one species of animal
into another species. This works because the antibodies produced by different species are different enough from
each other that they will provoke an immune response. For example, if you want a secondary antibody that will
recognize a human primary antibody, inject human antibodies into an animal like a rabbit. After the rabbit
immune response, the rabbit serum will contain antibodies that recognize and bind to human antibodies.
Secondary antibodies are frequently labeled to make them visible. In this experiment, the secondary antibodies
you will be working with are conjugated to an enzyme named horseradish peroxidase (HRP); HRP in the
presence of its substrate, TMB, produces a blue color.

62
Antigen

A. Structure of IgG bound to the HIV capsid protein p24 as determined by the X-ray crystallography
(Harris et.al. 1998, Momany et. Al. 1996). These structures can be downloaded from the Protein
Data Bank (www.pdb.ufrng br.(Berman et.al. 2000) using the PDB identification codes 1lGY and
1AFV and manipulated using free online software such as Rasmol and Protein Explorer.
B. A commonly used representation of an antibody bound to an antigen.

Controls in Immunoassays
For any immunoassay to be valid, it must include both positive and negative controls, i.e., samples that will
give known results. Controls are always run side by side with experimental samples. If you do not run a positive
control and the experiment gives negative results, how can you be sure the results are truly negative? What if
the assay simply did not work? If a positive sample gives a negative assay result, it is called a false negative.
Conversely, if you do not run a negative control and the experiment gives all positive results, how can you be
sure the results are truly positive? What if the assay was contaminated with antigen? If a negative sample gives
a positive assay result, it is called a false positive. Controls are also needed to guard against experimental error
and to ensure that the assay is working correctly. There can be problems with reagents, which can degrade due
to age or poor storage conditions. Operators can make mistakes by choosing the wrong reagents, making errors
in dilutions or in pipetting, or failing to remove unbound reagents. Poor record keeping is another source of
false assay results. Most of these possibilities can be checked for within the assay with the appropriate controls.
Now let’s put this all together.

The main steps in this antigen detection ELISA are: (refer to figure on the next page)

1. Add your sample and control samples to the wells in a microplate strip. Your samples contain many
proteins and may or may not contain the antigen. Incubate for 5 minutes to allow all the proteins in the
samples to bind to the plastic wells via hydrophobic interaction. This is called an immunosorbent assay
because proteins adsorb (bind) to the plastic wells.
2. Add primary antibody to the wells and incubate. The antibodies will seek out the antigen from the
many proteins bound to the well. If your sample contains the antigen, the antibodies will bind it tightly
and remain in the well.
3. Detect the bound antibodies with HRP-labeled secondary antibody. If the primary antibodies have
bound to the antigen, the secondary antibodies will bind tightly to the primary antibodies.
4. Add enzyme substrate to the wells, wait 5 minutes, and evaluate the assay results. If the antigen was
present in your sample, the wells will turn blue. This is a positive diagnosis. If the wells remain
colorless, the antigen was not present in your sample and the diagnosis is negative.

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 1

Procedure

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 7

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Exercise 9 Questions: Name__________________________
Section # _________________

Results
1. Which sample, A OR B contained the antigen for HIV?

Questions

2. What does ELISA stand for?

3. Why are enzymes used in this immunoassay? What is the enzyme used? What is its substrate? What
happens to TMB when the enzyme interacts with its substrate?

4. How are antibodies that are used in ELISA made?

5. Why do you need to assay positive and negative control samples as well as your experimental samples?

6. The samples that you will add to the microplate strip will contain many proteins and may or may not
contain the antigen. What will happen to the proteins in the plastic well if the sample contains the
antigen? What if it does not contain the antigen?

7. Why do you need to wash after every step?

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8. What reasons could there be for a negative result when the antigen is actually present?

9. Why do you assay your samples in triplicate?

10. What antibody-based tests can you buy at your local pharmacy?

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 Exercise 10
Chemical Control of Microbial Presence

Assessing Antimicrobial Effectiveness: The Disk Diffusion Method

Antimicrobials are chemical agents used in the treatment of infectious disease. Most antimicrobials work by
destroying microbes (microbiocidal) or by inhibiting their growth (microbiostatic). Naturally occurring
antimicrobials, produced by bacteria or fungi, are called antibiotics. Antibiotics that are modified in the
laboratory are called semi-synthetic antimicrobials. And antimicrobials that are completely synthesized in a
laboratory are called synthetic antimicrobials.

During the first part of the 20th century, there was hope that antimicrobials might lead to the elimination of
infectious disease. This hope was tempered by the emergence of pathogens resistant to antimicrobials. The
development of antimicrobial resistance led to the necessity for microbiology laboratory testing of a patient’s
pathogen against various antimicrobials (at various concentrations) to determine susceptibility or resistance
prior to treatment.

One laboratory method for determining the relative susceptibility of bacteria to antimicrobials is called the disk
diffusion method. In this method, an agar plate is seeded with the bacterium to be tested at an inoculum level
what will ensure confluent growth or a lawn of the bacterium after incubation. Filter paper disks that have been
impregnated with various antimicrobials at specific concentrations are then placed on the surface of the agar
plate. The plates are incubated during which time each antimicrobial diffuses into the agar surrounding its disk.
If the bacterium is killed or inhibited by the antimicrobial, there will be no growth in the area immediately
surrounding the disk (the so-called zone of inhibition). The relative susceptibility of a bacterium to the
antimicrobials tested can be assessed by comparing the sizes of the resulting “zones of inhibition.”

The disk diffusion method allows one to determine if a bacterium is sensitive to an antimicrobial (a zone of
inhibition will be present) at the concentration tested or is completely resistant to an antimicrobial (no zone of
inhibition will be present) at the concentration tested. It also allows one to determine if bacteria resistant to the
antimicrobial are emerging – this will be indicated by the presence of colonies (satellite colonies) growing within
a zone of inhibition. However, in the real world, a disk diffusion assays is just a starting point in the selection of
an antimicrobial to treat a bacterial infection.

In order to choose the most appropriate antimicrobial to treat an infection, a clinician needs to know the
minimum inhibitory concentration (MIC) or the lowest concentration of an antimicrobial that prevents visible
growth of a bacterium. The disk diffusion method does not provide this information. Instead, an assay called a
broth dilution assay, in which bacteria are inoculated into a liquid growth medium (a “broth”) in the presence
of a series of decreasing concentrations of an antimicrobial agent and the lowest concentration of antimicrobial
at which no growth is seen is determined, must be used.

You will utilize the disk diffusion method to compare the relative effectiveness of sixteen different antimicrobials
on six different bacterial cultures.

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Materials. (per lab group)

Various antimicrobials (see list at the end of this exercise) and dispenser
2 NA plates/culture (100 mm)
24-hour broth cultures (one per group) (instructors note that cultures might change):
Group 1 Staphylococcus aureus (S. aur.)
Group 2 Pseudomonas aeruginosa (P. aerug.)
Group 3 Escherichia coli (E. coli)
Group 4 Bacillus subtilis (B. sub.)
Group 5 Serratia marcescens (S. marc.)
Group 6 Enterobacter aerogenes (E. aero.)

Note to instructors: please ensure that the bacteria with which you were provided and the bacteria listed in the
tables are the same. Direct the students to make appropriate changes. Similarly, check that the antimicrobials in
the disk dispensers correspond with those listed in the table and direct the students to make any necessary
changes.

Procedure.

1. Obtain 2 sterile nutrient agar plates. Prepare your cultures by producing a microbial lawn. Label as
demonstrated by your instructor.

2. Dispense the antimicrobials as demonstrated by your instructor. Use a pair of forceps to gently press
disks onto the agar surface.

3. Invert the plates and incubate for 24 to 48 hours at 37oC.

Data Collection.

1. Measure the zone of inhibition for each antimicrobial. Record your data in Table 1. You will need to share
your group’s data with the other lab groups in your class, or enter it into the course Excel file, as directed by
your instructor.

In the space below Table 1, there is a section for you to note the presence of satellite colonies. These are colonies
that grow within a zone of inhibition and represent bacteria in the community that are resistant to the antibiotic
at the concentration being tested (your instructor will instruct you as to whether or not to include in Table 1 data
from antimicrobials where satellite colonies are present).

2. Calculate average zones of inhibition (mm) for each of the antimicrobials. Also, calculate the average zone of
inhibition for each of the bacteria. Refer to these averages to answer the questions that follow.
Note: if class data is compiled in Excel by your instructor, Excel will calculate the averages and ONLY the
averages need to be entered for data from other groups.

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 Table 1. Antimicrobial Data

Antimicrobial S. aur. P. aerug. E. coli B. sub. S. marc. E. aero. Ave. Zone of

inhibition
Antimicrobial
(mm)
Plate A (Antimicrobial 1)

1 Ampicillin (10mcg, AM 10)

2 Neomycin (5 mcg, N 5)

3 Tobramycin (10 mcg, NN 10)

4 Carbenicillin (100 mcg, CB 100)

5 Penicillin G (10 units, P 10)

6 Amikacin (30 mcg, AN 30)

7 Tetracycline (30 mcg, TE 30)

8 Amoxicillin (20 mcg, AMC 20)

Plate B (Antimicrobial 2)

9 Sulfamethoxazole/Trimethoprim( SXT)

10 Streptomycin (10 mcg, S 10)

11 Erythromycin (15 mcg, E 15)

12 Vancomycin (30 mcg, VA 30)

13 Bacitracin (10 units, B 10)

14 Chloramphenicol (30 mcg, C 30)

15 Oxacillin (1 mcg, OX 1)

16 Polymyxin B (300 units, PB 300)

Ave. Zone of Inhibition Bacteria

(mm)

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 Table 2. Antiseptics/Disinfectants Data

Antiseptics/Disinfectants S. aur. P. aerug E. coli B. sub. S. marc. E. aero. Ave. Zone of inhibition
Antiseptics/Disinfectants
(mm)
Plate C
(Antiseptics/ Disinfectants)
1 0.2 M Zinc Sulfate ZnSO4*7H2O

2 20% Cupric Sulfate CuSO4 *5H2O

3 0.1 M Mercuric Chloride HgCl2

4 0.2 M Silver Nitrate AgNO3

5 70% Ethanol (C2H5OH)

6 2% Sodium Azide

7 3% Hydrogen Peroxide (H2O2)

8 Betadine Sol. (10% Povidone-Iodine)

Plate D
(Antiseptics/ Disinfectants)
9 Micro-Quat Germicidal Detergent

10 Ci-Decon

11 Bleach

12 Anti-septic Hand Cleanser

13 Antibacterial Toilet Bowl Cleaner

14 Cepacol

15 Listerine

16 Scope

Ave. Zone of Inhibition

Bacteria (mm)

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Exercise 10 Questions. Name _________________________
Section # ______________________

Record the presence of any satellite colonies here. Note the antimicrobial and the bacterium (refer to list of
bacteria at beginning of lab and note full Genus and species name of bacteria).

1. Describe the zone of inhibition and how it can be used to assess antimicrobial sensitivity. Include a discussion
of the significance of satellite colonies.

2. Explain minimal inhibitory concentration (MIC)? Can MIC be determined using the disk diffusion method?
If not, then what method is used to determine MIC?

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3. Assume the chemical with the largest average zone of inhibition is the most effective of those tested. Complete
this table by referring to the class data in Tables 1 and 2:

Most effective antimicrobial

Least effective antimicrobial

Most effective antiseptic/ disinfectant

Least effective antiseptic/ disinfectant

4. Assume a bacterium shows a small zone of inhibition or none at all because it is resistant to the chemical
tested. Complete this table by referring to Tables 1 and 2. Please refer to list of bacteria at beginning of lab and note
full Genus and species name of the bacteria.

Bacterium most resistant to antimicrobials tested.

Bacterium least resistant (most sensitive) to

antimicrobials tested.

Bacterium most resistant to antiseptics/

disinfectants tested.

Bacterium least resistant (most sensitive) to

antiseptics/ disinfectants tested.

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