Microbiology 208-Laboratory Session Benjamin S. Weeks, Ph.D.

Laboratory Manual
Microbiology 208-Laboratory Sections
Adelphi University
Prepared by Benjamin S. Weeks, Ph.D.
Professor of Biological Sciences

MODELING NOSOCOMIAL
INFECTION
A laboratory manual uniquely focused on
understanding and preventing nosocomial infection in
the Nursing and Allied Health Industries.
Designed for HyFlex Presentation in the times of
CoVid-19

Student Name: .

Date and Time: .

Semester and Year: .

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Microbiology 0105-208-Laboratory Sections Benjamin S. Weeks, Ph.D.

ADELPHI UNIVERSITY LABORATORY SAFETY STATEMENT

Below is the Laboratory Safety Statement from the American Society for Microbiology (ASM),
Guidelines for Biosafety in Teaching Laboratories. The ASM recommends that this safety
statement be adapted as the University Laboratory Safety Statement for this course, Microbiology
0105-208-Laboratory sections. (https://www.asm.org/ASM/media/Education/Biosafety-Guidelines-
Appendix.pdf)
“The lab exercises in this course involve the use of living organisms. Although the microorganisms we
use are not considered to be highly virulent, all microorganisms should be treated as potential
pathogens (organisms capable of causing disease).
The following rules must be observed at all times to prevent accidental injury to and infection of
yourself and others and to minimize contamination of the lab environment:
1. Never place books, backpacks, purses, etc., on bench tops. Always place these in the assigned
cubicles. Keep manuals and pens on pull-out desks.
2. Electronic devices should not be brought into the lab. This includes, but is not limited to iPods, MP3
players, radios, cell phones, and calculators.
3. Clean your work area with dilute bleach solution at the beginning AND end of each lab.
4. Wash your hands with soap and dry with paper towels when entering and leaving the lab.
5. Wear a lab coat at all times while working in the lab to prevent contamination or accidental staining
of your clothing.
A.Closed-toe shoes (no sandals) are to be worn in the lab.
B.Long hair must be tied back to prevent exposure to flame and contamination of cultures.
C.Gloves should be worn when staining microbes and handling hazardous chemicals.
6. Do not place anything in your mouth or eyes while in the lab. This includes pencils, food, and
fingers. Keep your hands away from your mouth and eyes.
A.Eating and drinking are prohibited in the lab at all times.
B.This includes gum, cough drops, and candy.
C.Do not apply cosmetics in the lab. This includes Chapstick and Blistex.
D.Never pipet by mouth. Use a mechanical pipetting device.
7. Do not remove media, equipment, or bacterial cultures from the laboratory. This is absolutely
prohibited and unnecessary.
8. Do not place contaminated instruments such as inoculating loops, needles, and pipettes on bench
tops. Loops and needles should be sterilized by incineration, and pipettes should be disposed of in
designated receptacles of bleach solution.
9. Carry cultures in a test tube rack when moving around the lab or when keeping cultures on bench
tops for use. This prevents accidents and contamination of your person or belongings.

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10. Immediately cover spilled cultures or broken culture tubes with paper towels and then
saturate them with disinfectant solution. Notify your instructor that there has been a spill. After
15 minutes, dispose of the towels and broken items as indicated by your instructor.
11. Report accidental cuts or burns to the instructor immediately.
12. At the end of each lab session, place all cultures and materials in the proper disposal area.
13. Persons who are immune-compromised (including those who are pregnant or may become
pregnant) and students living with or caring for an immune-compromised individual are advised to
consult with your physician to determine the appropriate level of participation in the lab. Should your
physician determine that you should not participate in this lab, please have him or her write a note
stating the concerns. Alternative accommodations may be indicated.

OSHA INFORMATION
Material Safety Data Sheets (MSDS) are located ________________.
The first aid kit is located ____________________.
The eyewash station is located _________________.
The shower is located _________________________.
The fire extinguisher is located _______________________.

STUDENT AGREEMENT ON LABORATORY SAFETY

(Student Copy)
I have read the Laboratory Safety Statement of the Department of Biological Sciences, Adelphi
University, for course 0105-208-Laboratory Sections and I understand its content. I agree to abide by
all laboratory rules set forth by the instructor and stated in the laboratory manual. I understand that my
safety is entirely my own responsibility and that I may be putting myself and others in danger if I do
not abide by all the rules set forth by the instructor.

NAME OF STUDENT (PRINT): ________________________

SIGNATURE OF STUDENT: ________________________

DATE: ________________________”

The above student agreement is duplicated on the last page of the laboratory manual, please sign
both, keeping the above copy in the manual (student record) and removing the signed last page
of the manual and handing it in (University record).

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Microbiology 208-Laboratory Drawer Inventor and Work Flow Benjamin S. Weeks, Ph.D.

Microbiology 208-Laboratory Section Drawer Inventory

Each Drawer in room 24 should have the following stored in it for Microbiology 208.

Drawer Inventory

1) Ruler
2) Staining Tray
3) Staining Rack
4) Bibulous Paper
5) Lens Paper
6) Tungsten Loop
7) Clamp
8) Bunsen Burner
9) Striker
10) Sharpee or Wax Pencil

With regard to material needed routinely for laboratory safety, during all laboratory procedures,
students are required to wear gloves, laboratory coats and goggles, which will be provided. Students
are also required to clean their laboratory bench before and after each laboratory session and
disinfectant and paper towels will be provided.

Microbiology Session Work Flow

There are 14 Laboratory Sessions (Laboratory Sessions 1 – 14) and each has a note to the student,
purpose, background, materials, procedures, results and a discussion question assignment section.

I) Laboratory Opening: The Teaching Assistant is expected to open the door at the time of each
laboratory session start. Students will store their items and then go to their assigned benches. The
Teaching Assistant will then take attendance.

II) Laboratory Start: The instructor will direct the students to take 10 minutes to read the
laboratory thoroughly. Reading ahead can be assigned but here it is considered best to read
immediately prior to the work.

III) Laboratory Introduction: The instructor will present and review all sections of the Laboratory
Session for the day. These include the note to student purpose, background, materials and methods,
procedures (best drawn out on board), how to document the results, and the discussion questions.

IV) Laboratory Work: Students perform and document work.

V) Laboratory Close: Students may finish work at slightly different times. When done the
student is to clean their bench top and work space appropriately and then check-out with the Teaching
Assistant who will both confirm the student’s attendance and also check that the student completed
their work and cleaned their bench. The Teaching Assistant is then responsible for cleaning the med-
path waste in the laboratory, cleaning common work stations, cleaning the cubbyholes, turning out the
lights, reviewing that the gas is off and the laboratory orderly and neat for the next session. The
Teaching Assistance will then be the last to leave the room.

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 Laboratory Session 1
Introduction to the Microbiology 208 - Laboratory
Note to the student: Before the Professor begins, you will be given an opportunity and will be
expected to read the laboratory for the day. At the end of most laboratory sessions are a set of
questions which you are responsible for. Please read these questions before the professor begins.
The answers to most of these questions will either be in the laboratory manual reading or written
on the board as the professor presents the laboratory purpose and background, so it is best that
you take notes on the laboratory introductory lectures so you will have a convenient source of
notes to both complete the manual questions, but also for the laboratory exams. Note that at the
end of this and each laboratory is space designated for each question where you can both a) take
notes and b) put final submitted answers. Further much of this material will be the subject of
two laboratory examinations and notes taken during class may be invaluable when studying for
these exams.

PURPOSE
1) To introduce the teaching assistant
2) To review laboratory safety practices
3) To discuss the course structure (manual, syllabus, grading) of the Laboratory Section of
Microbiology 0105-208
4) To discuss the issue of nosocomial infection in healthcare.
5) To visit the www.cdc.gov website and review the data and statistics regarding
nosocomial infection in the United States and to also show students where the CDC site
has a section committed to readings on the sources, causes and fixes to nosocomial
infection as well as guidelines and projected goals.
6) To review laboratory drawer inventory and complete the inventory of any drawers
lacking items.

BACKGROUND
Teaching Assistant: The teaching assistant will open the laboratory doors at the start of each
session. Students are not to enter the laboratory if the teaching assistant or professor is not
present. If the door is open prior to the start of class, the student is not to enter the laboratory
until the start of each session. The student may ask the teaching assistant questions on the
content of the laboratory as well for help in finding or identifying materials. The teaching
assistant also has authority regarding student safety and behavior in the laboratory as well and
safe laboratory practices.
Laboratory Safety Practices: This laboratory will involve the use both bacterial the student
cultures from the environment which will be kept sealed and also a variety of known
nonpathogenic bacteria provided by the instructor. Never the less, when working with bacteria,
1
it is important to maintain strict safety precautions. Therefore when working in the laboratory
students will be required to wear laboratory coats, latex or nitrile gloves, and goggles. With
regard to material needed routinely for laboratory safety, during all laboratory procedures,
students are required to wear gloves, laboratory coats and goggles, which will be provided.

2
Items such as backpacks, books and laptops are not to be brought to the laboratory bench. Upon
entering the laboratory all items other than your laboratory manual are to be stored in the nooks
upon entry to room 24. Other electronic devices such as cell phones and earbuds are also not to
be brought to the bench.
Shorts and open toed shoes are not allowed in the laboratory. You must not to wear baggy
clothing and if you have long hair you must tie it back behind your shoulders.
Food and drinks are not allowed through the door into the laboratory, nor is waste from food or
drinks or empty drink containers or food wrapping allowed in the laboratory or laboratory
garbage cans.
Clean your bench before and after the laboratory and wash your hands before and after the
laboratory.
Do not put anything into your mouth while in the laboratory and work with your hands below
your shoulders.
When finished working with materials, dispose of all items appropriately into the appropriate
waste containers as you are direct by your Professor.
Report all cuts, injury or burns during the laboratory to the Professor immediately.
Pages two and three of this laboratory manual is the American Society for Microbiology,
Guideline for Safe Practices in the Teaching Laboratory. The instructor will review the
guideline in entirety. At the end of the guideline is a Student Agreement on Laboratory
Safety which each student should sign (Student’s Copy) during the first laboratory session. The
students will be tested on the material and so the safety sheet should remain attached, so a second
Student Agreement on Laboratory Safety is attached to the end of this manual to be signed
and returned to the Professor or Teaching Assistant (University Copy).
Laboratory Section Course Structure: This is an introductory course with no prerequisites
and does not count for Biology Major credit and while the course is designed for Nursing
Students, the course is open to all students and there is no prerequisite. Please check with your
academic advisor to determine if this course is appropriate for you.
Assignments and Grades: There laboratory grade will be based on a) attendance, b) the
laboratory manuals, and c) laboratory exam performance. Once the laboratory grade is
determined, the grade will be submitted to the lecture professor who will use the laboratory grade
to determine the overall grade.
Absences: Absence is allowed for only medical or religious reasons. If the student has a
religious Holiday the student must inform the Professor on the first day of class the days that will
be missed. Medical absences are permitted with a medical note. If the student has an unexcused
absence, it likely the student will not be able to make up the work and unlike any dynamic in the
lecture section the student grade may immediately suffer.

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Notes and Content Study Materials: At the beginning of each laboratory time will be given to the
student to read the laboratory including the “Discussion Assignment Questions”. The Discussion
and Assignment Question section has a place designated for “notes” and “answers”. There is
also lined space provided for general notes. After time is given to reading, there will be a lecture
and introductory discussion on the background and topic material of the laboratory. Students are
expected to take note either in the Discussion Assignment Questions section specific to the
section or in the general notes section. While the topics covered in the Discussion Assignment
Questions are listed here in this manual and are also available in the textbook and at the CDC
website, students are expected to take notes down into the laboratory manual during the
Professor’s introductory lecture and discussion from which to study since the student will not
only answer the Discussion Assignment Questions, but will also be tested on the laboratory
material in two laboratory exams.
Online and email Communication: Students are expected to use the CLASS and Moodle system
and regularly check their Adelphi University email accounts for routine communication from the
professor and uploads of laboratory exams, assignments and articles when applicable.
Assigned seating: Students will select a bench seat which they are to remain at as an assigned
seat for the remainder of the semester. Each seat is associated with a cabinet and drawer number.
This will be the assigned seating number and attendance is best take with this numbering system.
Nosocomial Infection: The word Nosocomium in Latin means hospital. Infections acquired
while in the hospital or in any clinical setting is known as a nosocomial infection or a hospital
acquired infection (HAI). The approximate nosocomial infection rate in the USA is 7.5% or
1/14 patients. There are 100,000 death per year in the USA due to nosocomial infection and with
3,000,000 nurses in the work force that’s 1/30 death/nurse/year due to a nosocomial infection or
1 death/nurse/30 year career. Students should understand that they will cause the death of a
patient die to nosocomial infection during the course of their career and that washing their hands
and keeping their hands below their shoulders while working in the clinical setting, they will
save lives. That what they learn in this laboratory may be mundane but it will be a life or death
lesson that they learn.
The professor will open the www.cdc.gov website and on the website search HAIs (Hospital
Aquired Infections). This search will offer a link to https://www.cdc.gov/hai/. There are 9 tabs
that deal with HAIs labeled as follows. These topics will be explored and discussed during this
laboratory period. They are
-Types of Healthcare-associated Infections
-HAI Data and Statistics
-Guidelines & Recommendations
-Patient Safety: What You Can Do to Be a Safe Patient
-Containment Strategy Responding to Emerging AR Threats
-Preventing Healthcare-associated Infections
-State-based HAI Prevention
-Innovative Research to Support Safe Healthcare
-Laboratory Resources

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MATERIALS
None
PROCEDURES
None
RESULTS
None
DISCUSSION ASSIGNMENT QUESTIONS (To be completed by student during available time
during the laboratory session and/or outside of the scheduled laboratory course session meeting if time requires. All
questions for all laboratories must be completed before handing in the manual in order to receive full credit. Manuals are
due after the 14th (last) laboratory session meeting in the last week of the semester.)

1) What is the rate of nosocomial infection in the United States?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

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 Laboratory Session 2

A)Collection of Bacteria from the Environment and Modeling

Nosocomial Infection

B) Colony Morphology

Part A) Modeling Nosocomial Infection

Note to the Student: Today you will both collect bacteria from the environment and also start a
culture of mixed known bacteria. Both the environmental sample and the sample you mix today
will contain mixtures of bacteria. The ones from the environment will be unknown, so we don’t
want to risk working with them extensively. They do however represent a real mixture of
bacteria that could cause nosocomial infections and the mixture of different bacterial types
reflects the reality of exposure in the hospital setting. Therefore we will create a mixture of
bacteria know to be nonpathogenic to humans to model the real mixture collected from the
environment. In both cases the plates will represent patients. Please take time to read the entire
laboratory before we begin. At the end of most laboratory sessions are a set of questions which
you are responsible for. Please read these questions before the professor begins. The answers to
most of these questions will either be in the laboratory manual reading or written on the board as
the professor presents the laboratory purpose and background, so it is best that you take notes on
the laboratory introductory lectures so you will have a convenient source of notes to both
complete the manual questions, but also for the laboratory exams. Note that at the end of this
and each laboratory is space designated for each question where you can both a) take notes and
b) put final submitted answers. Further much of this material will be the subject of two
laboratory examinations and notes taken during class may be invaluable when studying for these
exams.

PURPOSE
1) To demonstrate that there are bacteria on nearly all surfaces in a hospital
2) To demonstrate that the bacteria on hospital surfaces cause nosocomial infection
3) To demonstrate by colony morphology that the bacteria patients are exposed to in the
hospital are mixtures of many different types of bacteria
4) To identify and distinguish bacteria in a mixture of five known BSL1 micro-organisms
based on colony morphology
5) To show and document that the mixed real world environmental sample is modeled by
the BSL1 environmental sample based on range of colony characteristics

BACKGROUND

“Microorganisms are found throughout the environment: in the air and water; on the surface of
6
objects, clothes, tables, floors; in soil and dust; and on the skin and mucous membranes of your
bodies. These widely present microorganisms ordinarily are of no concern to healthy humans,
provided we maintain good hygiene in our daily living. In hospitals, however, where susceptible
patients must be protected from hospital-acquired infections, the concentration and distribution
of microorganisms in the environment are of great importance. Frequent monitoring of the
environment is one of the responsibilities of the hospital epidemiologists, who may be a
microbiologist, nurse, or physician. During patient care all environments and equipments are
sterilized but what happens in between patients to these sterile surfaces?” (from:
https://biocyclopedia.com/index/microbiology_methods/basic_techniques_biotechnologies/
culturing_microorganisms_from_environment.php)

MATERIALS
Each student needs:
1) Three nutrient agar plates
2) Three sterile cotton swabs
3) Access to a mixed culture of:
Bacilus subtilus
Clostridium sporenges
E. Coli
Seratia marcescens
Staphylococcus epidermidis

PROCEDURES
1) Label the agar side of all three plates with the date and your initials. Always label agar
plates on the agar side
2) Label two plates Env. and the third plate BSL1 Env.
3) Take the two plates labeled Env, a marker and two cotton swabs and leave the laboratory,
room 24.
4) Imagine you are a nurse in a hospital, the cotton swabs are your fingers or a devise and
the nutrient agar plates are two of your patients.
5) Select a surface and imagine it to be a surface in a hospital
6) Rub the cotton swab on the surface 30 times and then using the same area of the swab,
serpentine across the plate exposing the nutrient agar to the part of the swab exposed to
the surface as shown below:

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 7) Imagine that this is you exposing your patient to hospital born bacteria.
8) Label the plate with the location you sampled and using your second plate and cotton
swab, repeat. You can just write down 1 and 2 on the plate for locations by recording the
locations for 1 and 2 in the table in the Results section below.
9) Return plates to tray for incubation at 37oC
10) Gently vortex the BSL1 mixed culture and dip you last cotton swab into the mixture and
as shown above, serpentine across your third plate labeled BSL1 Env and return the plate
to the teaching assistant for incubation at 37oC
11) The plates will be incubated for 24 hours and placed in a 4oC refrigerator until you return
next week to continue your work. This step will be done for you.

RESULTS
You will not have results yet, however the open space below is a good place to jot down the two
environmental locations you sampled.

Plate Environmental Location

1

Part B) Colony Morphology

Note to the Student: Today you will examine the colonies that have grown on your plate.
Because the plate represents your patient, these colonies represent the nosocomial infection, you
will review infections that you can cause to your patient. The environmental plates will be
discarded and left unopened, because it is possible that these plates contain real human
pathogens. The plate with the know non-pathogenic organisms will be compared to the

8
environmental and subcloned and continued to be worked with throughout the semester as a
model of nosocomial infection. Please read the discussion questions before the professor begins.
The answers to most of these questions will either be in the laboratory manual reading or written
on the board as the professor presents the laboratory purpose and background, so it is best that
you take notes on the laboratory introductory lectures so you will have a convenient source of
notes to both complete the manual questions, but also for the laboratory exams. Note that at the
end of this and each laboratory is space designated for each question where you can both a) take
notes and b) put final submitted answers. Further much of this material will be the subject of
two laboratory examinations and notes taken during class may be invaluable when studying for
these exams.

PURPOSE
1) To demonstrate that there are bacteria on nearly all surfaces in a hospital
2) To demonstrate that the bacteria on hospital surfaces cause nosocomial infection
3) To demonstrate by colony morphology that the bacteria patients are exposed to in the
hospital are mixtures of many different types of bacteria
4) To identify and distinguish bacteria in a mixture of five known BSL1 micro-organisms
based on colony morphology
5) To examine plates for bacterial growth
6) To practice identifying the common characteristics of bacterial colonies
7) To characterize the colonies of bacteria found from the environment

BACKGROUND
Last week samples were collected to establish the model of nosocomial infection. Once again,
the cotton swab represented a nurse’s finger or a device such as a catheter, the Adelphi
University science building hallways represented the hospital, the nutrient agar plate represented
the patient and the bacteria now growing on the nutrient agar represents the nosocomial
infection.

Normally bacteria are cultured in pure isolated cultures. This is required for research scientist
determine antibiotic sensitivity to specific bacteria and help to discover antibiotics that will kill
specific populations of bacteria such as pathogenic strains or those that have developed drug
resistance. By sampling the environment, we have collected multiple types of bacteria, so we
are not able to do a normal experiment with these mixed isolates, however, these mixed
environmental samples more accurately reflect the real world and therefore more accurately
models the real world. The environmental samples must remain sealed for biosafety reasons,
however the plate with the mixed BSL1 environmental sample can be opened and worked with.
Colonies and biofilms are both populations of cells, however colonies are populations of micro-
organisms that are derived from the same original cell. In this regard, the cells of a colony are
clones. While some of the students may have isolated colonies on some of their plates, we
cannot be sure these are true colonies and most students are likely to see large areas of bacteria
growing with no clear boundary between colonies. In essence most students will have a biofilm
of bacteria growing, or several biofilms of mixed bacterial types. When bacteria grow in and on
surfaces in the body they are not seen in colonies there either, but rather are mixed populations

9
and in biofilm form, such as is seen with plaque. Biofilms are often seen to form on stints and
implants or catheters in cases of nosocoomial infection.

Unlike biofilms, individual colonies have distinct characteristics such as form, margin, elevation,
pigmentation, opacity and others. While mixtures and biofilms are not colonies we can see some
parallel common and perhaps remnant colony characteristics in the biofilms. Students will
examine the biofilms and mixed cultures for “colony-like” characteristics.

These plates do not have an immune system. However patients in the hospital can have
weakened immune systems making them more like the agar plate. The natural immune state of
the patient can affect their susceptibility to infection, along with drugs with a wide range of
purposes which will lower immunity. Patients at a higher risk of infection include the young, the
elderly and the immunodeficient. The young are susceptible because the immune system is
adaptive. Passive immunity is offered by the maternal system. Elderly experience atrophy of the
Thymus when T-cell mature and loss of hematopoetic bone marrow. Further, in addition to age,
natural immunity is affected in cases of genetic immundeficiencies as well as conditions such as
AIDS. Drugs can also reduce immunity. Cancer grows rapidly like cells of the immune system
so cancer drugs target the cell cycle. Due to grafts versus host disease, transplant patients are on
immunosuppressive drugs for life as are patients with autoimmunity. Wounds and burns are also
at high risk for infection due to the damage to and circumvention of aspects of the innate
immune system that goes along with tissue damage.

Bacteria are all around you, yet we are unable to see them with the naked eye.
When looking at your plates you can see the bacterial colonies, this means you are looking at
billions of bacterial cells! As the bacterial cells grow and divide, colonies begin forming on
your plate. A colony is a result of a single bacterial cell dividing multiple times during our
incubation period. When you see many bacteria crowded together it is known as confluent
growth. You may also notice colonies of bacteria that are more spread out.
Scientists may use observations of the shape, size and appearance of a bacterial colony
to identify it and characterize it by a description known as colony morphology.

The below is from:

https://www.sciencebuddies.org/science-fair-projects/references/interpreting-agar-plates

“Common details to observe and note about bacterial colonies are as follows:

 Form – What is the basic shape of the colony? For example, circular, filamentous, etc.
 Elevation – What is the cross sectional shape of the colony?
 Surface – How does the surface of the colony appear? For example, smooth, glistening,
rough, dull (opposite of glistening), rugose (wrinkled), etc.
 Opacity – For example, transparent (clear), opaque, translucent (almost clear, but
distorted vision, like looking through frosted glass), iridescent (changing colors in
reflected light), etc.
 Pigmentation – For example, white, buff, red, purple, etc.

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 ”

Above From: https://www.sciencebuddies.org/science-fair-projects/references/interpreting-agar-

plates

MATERIALS
1) 3 nutrient agar plates from previous lab.

PROCEDURES
1. Obtain sample plates from last week
2. Record the location where you obtained the samples.

Plate Environmental Location

1

3. Complete the attached charts. There is a chart for each environmental

sample location.
4. Locate the different colony types you find on each plate (bacterial growths
that appear to have different morphologies) and number the different types
on your chart [i.e. A clump a white, shiny, circular bacteria could be called

11
 “colony grouping #1” and yellow, dull, filamentous bacteria could be
called “colony grouping #2”]
5. Count the number of colonies for each type. Make sure to note this under
“Number of Colonies”
6. For the same colony, fill out the remainder of the chart (i.e., form,
elevation, margin, etc.)
7. Repeat this process for all your colony types

**If colonies are too many to count, fill in TMD (Too Many to Determine) in the designated
area on chart**

*Ask instructor if you are having trouble determining certain morphologies.

RESULTS
Complete tables below

Or remote view:

https://www.smithsonianmag.com/smart-news/what-happens-when-you-culture-bacteria-eight-
year-olds-hand-180955528/

https://commons.wikimedia.org/wiki/File:VCU_agar_plate_colonies.jpg

https://www.researchgate.net/figure/Positive-control-group-showing-bacterial-colonies-
incubated-from-the-researcher-arm-on_fig4_312222937

https://www.123rf.com/photo_44756574_yellow-bacterial-colonies-on-a-petri-plate-dish-
isolated-on-a-black-background-pure-oil-degrading-ba.html

https://www.123rf.com/photo_45336374_orange-bacterial-colonies-on-a-petri-dish-agar-plate-
isolated-on-a-black-background-pure-oil-degradi.html

12
13
DISCUSSION ASSIGNMENT QUESTIONS
Questions for Lab 1 – 2
14
 1) What is the rate of nosocomial infection in the United States?

2) . Compare and contrast a colony with a biofilm. Give examples of a biofilms in the
human body. Define both terms.

3) Name five different ways a colony can be characterized.

4) What is agar? Does it have nutritional value?

Laboratory Session 3 +4 (In Class/Two sessions)

A)How Clean is the Environment?

B) Use of Microscope

Part A) A)How Clean is the Environment?

PURPOSE
1) To see why it is so important to have a clean working environment when working with
patient samples.
2) To clean working bench surface with disinfectants and monitor growth of bacteria right
after sterilizing and after a certain period of time

BACKGROUND
Last week samples were collected to establish the model of nosocomial infection. Once again,
the cotton swab represented your finger or a device such as a catheter, the Adelphi University
15
science building hallways represented the hospital, the nutrient agar plate represented the patient
and the bacteria now growing on the nutrient agar represent the nosocomial infection.

The below is from:

https://biocyclopedia.com/index/microbiology_methods/basic_techniques_biotechnologies/
culturing_microorganisms_from_environment.php
“Microorganisms are found throughout the environment: in the air and water; on the surface of objects,
clothes, tables, floors; in soil and dust; and on the skin and mucous membranes of your bodies. These
widely present microorganisms ordinarily are of no concern to healthy humans, provided we maintain
good hygiene in our daily living. In hospitals, however, where susceptible patients must be protected from
hospital-acquired infections, the concentration and distribution of microorganisms in the environment are
of great importance. Frequent monitoring of the environment is one of the responsibilities of the hospital
epidemiologists, who may be a microbiologist, nurse, or physician. During patient care all environments
and equipments are sterilized, but what happens in between patients to these sterile surfaces?”
https://biocyclopedia.com/index/microbiology_methods/basic_techniques_biotechnologies/
culturing_microorganisms_from_environment.php

MATERIALS
 Nutrient Agar Plates (contains nutrients necessary for bacteria to grow)
 Sterile Swabs
 Disinfectant Cleaner
 Sterile Water Test Tubes
 Clock
 Markers or Wax Pencils
 Latex Gloves

PROCEDURES
1. Obtain three (3) Nutrient Agar Plates
o Label as follows:
 Name
 Work Bench Number
 Date
 Lab #
 Type of Disinfectant used
 Label one plate with 0 minutes, the second with 30 minutes, and
the third with 90 minutes.
2. Choose one of the available disinfectants (such as 95% ethanol or Disinfectant
Cleaners) and wipe clean your work bench with the paper towels. Make sure you
clean all of your work area.
3. Obtain three (3) Sterile Swabs (one for each time/plate designation).
4. Open one of the sterile swab wrappers from the end opposite the cotton swab (so
you don’t contaminate the cotton swab with the possible bacteria on your gloves).
5. Pull out the swab from the wrapper and place it into the water in one of the test
tubes to wet the swab. (do not worry about the varying quantity of water in the
test tubes, as long as the swab is moist you are fine)
16
 6. Swab your bench surface (make sure you do not swab areas you did not clean)
7. Take the nutrient agar plates labeled 0 MINUTES and streak the plate with your
cotton swab. See the picture below for streaking technique.

8. After 30 minutes, repeat steps 3-6 for the nutrient agar plate labeled 30
MINUTES. Make sure to swab the area of the workbench where you
initially swabbed.
9. After 90 minutes, repeat steps 3-6 for the nutrient agar plate labeled 90
MINUTES. Make sure to use a fresh tube of sterile water and swab the same
surface of the work bench.
10. Place all three plates on the tray marked “incubation at 37 degrees” at the front of
the class.

Next week we will observe these plates for growth and further analyze them

RESULTS
Document next week
Analysis of Disinfectant Efficacy

Time Number of Colonies

0 minutes

30 minutes

90 minutes

DISCUSSION ASSAGNMENT QUESTIONS

17
1. What do you think will happen after the 30 minutes? Will there be bacterial growth
on the plate? Why?
NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

2. Why do you think it is so important to keep a clean working environment when

working with patient samples such as blood, urine, etc.?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

3. Why is it so important to wear gloves and also to change them from time to time
when working in a laboratory?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

18
Part B) Use of the Microscope
The Microscope
PURPOSE
 To study the compound microscope and learn:
 Its important parts and their functions
 How to focus and use it to study microorganism
 Its proper care and handling

BACKGROUND
A good microscope is an essential tool for any microbiology laboratory. There are many kinds of
microscopes, but the type most useful in diagnostic work is the compound microscope. By means of
a series of lenses and a source of bright light, it magnifies and illuminates minute objects such as
bacteria and other microorganisms that would otherwise be invisible to the eye. This type of
microscope will be used throughout your laboratory course. As you gain experience using it, you
will realize how precise it is and how valuable for studying microorganisms present in clinical
specimens and in cultures. Even though you may not use a microscope in your profession, a
firsthand knowledge of how to use it is important. Your laboratory experience with the
microscope will give you a lasting impression of living forms that are too small to be seen unless
they are highly magnified. As you learn about these "invisible" microorganisms, you should be
better able to understand their role in transmission of infection.
MATERIALS
 An assigned Microscope
 Lens paper
 Immersion oil
 Prepared slides

PROCEDURES
Important Parts of the Compound Microscope and Their Functions

1. Look at the microscope assigned to you and compare it with the photograph in figure
1.1. Notice that its working parts are set into a sturdy frame consisting of a base for
support and an arm for carrying it. (Note: When lifting and carrying the microscope,
always use both hands; one to grasp the arm firmly, the other to support the base (fig.
1.2). Never lift by the part that holds the lenses.)
2. Observe that a flat platform, or stage as it is called, extends between the upper lens
system and the lower set of devices for providing light. The stage has a hole in the
center that permits light from below to pass upward into the lenses above. The object
to be viewed is positioned on the stage over this opening so that it is brightly
illuminated from below (do not attempt to place your slide on the stage yet). Note the
adjustment knobs at the side of the stage, which are used to move the slide in vertical
and horizontal direction on the stage. This type of stage is referred to as a mechanical
stage.
19
3. A built-in illuminator at the base is the source of light. Light is directed upward
through the Abbe condenser. The condenser contains lenses that collect and
concentrate the light, directing it upward through any object on the stage. It also has
a shutter or iris diaphragm, which can be used to adjust the amount of light admitted.
A lever (sometimes a rotating knob) is provided on the condenser for operating the
diaphragm. The condenser can be lowered or raised by an adjustment knob.
Lowering the condenser decreases the amount of light that reaches the object. This is
usually a disadvantage in microbiological work. It is best to keep the condenser fully
raised and to adjust light intensity with the iris diaphragm.
4. Above the stage, attached to the arm, a tube holds the magnifying lenses through
which the object is viewed. The lower end of the tube is fitted with a rotating
nosepiece holding three or four objective lenses. As the nosepiece is rotated, anyone
of the objectives can be brought into position above the stage opening, the upper end
of the tube holds the ocular lens, or eyepiece (a binocular scope permits viewing with
both eyes through two oculars).
5. Depending on the brand of microscope used, either the rotating nosepiece or the stage
can be raised or lowered by coarse and fine adjustment knobs. These are located
either above or below the stage. On some microscopes they are mounted as two
separate knobs; on others they may be placed in tandem (see fig. 1.1) with the smaller
fine adjustment extending from the larger coarse wheel. Locate the coarse adjustment
on your microscope and rotate it gently, noting the upward or downward movement
of the nosepiece or stage. The coarse adjustment is used to bring the objective down
into position over any object on the stage, while looking at it from the side to avoid
striking the object and thus damaging the expensive objective lens (fig. 1.3). The fine
adjustment knob moves the tube to such a slight degree that movement cannot be
observed from the side. It is used when one is viewing the object through the lenses
to make the small adjustments necessary for a sharp, clear image.
6. Turn the adjustment knobs slowly and gently, as you pay attention to the relative
positions of the objective and object. Avoid bringing the objective down with the fine
adjustment while viewing, because even this slight motion may force the lens against
the object. Bring the lens safely down first with the coarse knob; then, while looking
through the ocular, turn the fine knob to raise the lens until you have a clear view of
the subject. Rotating the fine adjustment too far in either direction may cause it to
jam. If this should happen, never attempt to force it; call the instructor. To avoid
jamming, gently locate the two extremes to which the fine knob can be turned, then
bring it back to the middle of its span and keep it within one turn of this central
position. With practice, you will learn how to use the coarse and fine adjustment
knobs in tandem to avoid damaging your slide preparations.
7. The total magnification achieved with the microscope depends on the combination of
the ocular and objective lens used. Look at the ocular lens on your microscope. On
most microscopes you will see that it is marked “10X” meaning that it magnifies 10
times. Now look at the three objective lenses on the nosepiece. The short one is the
low-power objective. Its metal shaft bears a “10X” mark, indicating that it gives
tenfold magnification. When an object is viewed with the 10X objective combined
with the 10X ocular, it is magnified 10 times 10 or X 100. Among your three

20
 objectives, this short one has the largest lens but the least magnifying power. The
other three objectives look alike in length, but one is an intermediate objective, called
the high power (or high-dry) objective. It may or may not have a colored ring on it.
What magnification number is stamped on it? ________What is the total
magnification to be obtained when it is used with the 10X ocular? _________ The
fourth objective, which almost always has a colored ring, is called an iol-immersion
objective. It has the smallest lens but gives the highest magnification of the four.
What is its magnifying number? ________ What total magnification will it provide
together with the 10X ocular? ________ This objective is the most useful of the four
for the microbiologist because its high magnification permits clear viewing of all but
the smallest microorganism (viruses require an electron microscope). As its name
implies, this lens must be immersed in a drop of oil placed on the object to be viewed.
The oil improves resolution of the magnified image, providing sharp detail even
though it is greatly enlarged. The function of the oil is to prevent any scattering of
light rays passing through the lens. Notice that the higher the magnification used, the
more intense the light must be, but the amount of illumination needed is also
determined by the density of the object. For example, more light is needed to view
stained then unstained preparations.
8. The focal length of an object is directly proportional to the diameter of its lens. You
can see this by comparing your four objectives when positioned as close to the stage
as the coarse adjustment permits. First place the low-power objective in vertical
position and bring it down with the coarse knob as far as it will go (gently!). When
the object is in focus, the distance between the end of the objective, with its large
lens, and the top of the cover glass is the focal length. Without moving the coarse
adjustment, swing the next objective carefully into the vertical position, and note the
much shorter focal length. Swing the next objective carefully into the vertical
position, and again, note the much shorter focal length. Now, with extreme caution,
bring the oil-immersion objective into place, making sure your microscope will
permit this. If you think the lens will strike the stage or touch the condenser lens,
don’t try it until you have raised the nosepiece or lowered the stage (depending on
your type of microscope) with the coarse adjustment. The focal length of the oil-
immersion objective is between 1 and 2 nun, depending on the diameter of the lens it
possesses (some are finer than others). Never swing the oil-immersion objective into
use position without checking to see that it will not make contact with the stage, the
condenser, or the objects being viewed. The oil lens alone is one of the most
expensive and delicate part of the microscope and must always be protected from
scratching or other damage.
9. Take a piece of clean, soft lens paper and brush it lightly over the ocular and
objective lenses and the top of the condenser. With subdued light coming through,
look into the microscope. If you see specks of dust, rotate the ocular in its socket to
see whether the dirt moves. If it does, it is on the ocular and should be wiped off
more carefully. If you cannot solve the problem, call the instructor. Never wipe the
lenses with anything but clean, dry lens paper because solvents can damage the
lenses. Natural oil from eyelashes, mascara, or other eye makeup can soil the oculars
badly and seriously interfere with microscopy. Eyeglasses may scratch or be

21
 scratched by the oculars. If they are available, protective eyecups placed on the
oculars prevent these problems. If not, you must learn how to avoid soiling or
damaging the ocular lens.
10. If oculars or objectives must be removed from the microscope for any reason, only
the instructor or other delegated person should remove them. Inexperienced hands
can do irreparable damage to a precision instrument.
11. Because students in other laboratory sections may also use your assigned microscope,
you should examine the microscope carefully at the beginning of each laboratory
session. Report any new defects or damage to the instructor immediately.

Observing slides

1. Gently take the stained and prepared slide that has been provided (on the workbench).
2. Place the slide securely on the stage, making certain it cannot slip or move. Position
it so that light coming up through the condenser passes through the center of the
stained area.
3. Bring the low-power objective (40X) into vertical position and lower it as far as it
will go with the coarse adjustment, observing from the side.
4. Look through the ocular.
5. Adjust the two oculars horizontally to the width between your eyes until you have a
single, circular field of vision.
6. Now bring the objective slowly upward with the coarse adjustment until you can see
small, blue objects in the field. (Make certain the condenser is fully raised, and adjust
the light to comfortable brightness with the iris diaphragm).
7. Use the fine adjustment knob to get the image as sharp as possible.
8. Now use the stage adjustment knob to move the slide slowly around, up and down,
back and forth. The low-power lens should give you an overview of the preparation
and enable you to select an interesting are for closer observation at the next higher
magnification.
9. When you have selected an area you with to study further, swing the 100X objective
into place. If you are close to sharp focus, make your adjustment with the fine knob.
If the slide is badly out of focus with the new objective in place, look at the body tube
and bring the lens down close to, but not touching, the slide. Then, looking through
the ocular, adjust the lens slowly, first with the coarse adjustment, then with the fine,
until you have a sharp focus. Notice the difference in magnification of the structures
you see with this objective as compared with the previous one.
10. When you have focused the specimen at 100X, swing the 400X objective into place.
If you are close to focus, use the fine knob.

Do not continue without instructor approval…

11. Move the 400X lens a little to one side and place a drop of oil on the slide, directly
over the stage opening. With your eyes on the oil-immersion objective, bring it
carefully into position making certain it does not touch the stage or slide. Most

22
 microscopes are now parfocal; that is, the object remains in focus as you switch from
one objective to another. The fine adjustment alone will bring the object into sharp
focus. When you have a sharp focus, observe the difference in magnification
obtainable with this objective as compared with the other two. It is about 2 times
greater than that provided by the high power objective, and about 10 times more than
that of the low-power lens.
12. Record your observations by drawing in each of the following circles several of the
microbial structures you have seen, indicating their comparative size when viewed
with each objective.
13. When you have finished your observations, lower the stage or raise the objective
before removing the slide. Then remove the slide (taking care not to get oil on the
high-dry lens). Gently clean the oil from the oil-immersion
objective with a piece of dry lens paper.
14. Under each drawing, indicate the total magnification ™ obtained by each objective
combined with the ocular.

23
RESULTS

DISCUSSION ASSIGNMENT QUESTIONS

NOTE QUESTIONS IN READING

1. List the optical parts of the microscope. How does it achieve magnification? Resolution?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

2. What is the function of the condenser?

NOTES DURING LECTURE/DISCUSSION:

24
 FINAL SUBMITTED ANSWER:

3. What is the function of the iris diaphragm? To what part of the human eye would you
compare it?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

4. Why do you use oil on a slide to be examined with the oil-immersion objective?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

5. What is the advantage of parfocal lenses?

NOTES DURING LECTURE/DISCUSSION:

25
 FINAL SUBMITTED ANSWER:

6. If 5X, instead of 10X oculars were used with the same objectives now on your
microscope, what magnifications would be achieved?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

7. From reading in your textbook, can you name the two other types of microscopes? Is
their magnification range higher or lower than that of the compound light microscope?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

26
 Laboratory Session 5
Laboratory Exam 1
PURPOSE
To test students on the definitions and statistics regarding nosocomial infection, ways to
prevent hospital acquired infection, colonies, biofilms, use of agar plates, and laboratory
safety guidelines,

BACKGROUND
None
MATERIALS
None
PROCEDURES
None
RESULTS
None
DISCUSSION QUESTIONS
None

Topics to be covered on this Exam include, laboratory safety, nosocomial infection rates,
death rates due to nosocomial infection, how to fight nosocomial infection, types of
nosocomial infection, the definition of colonies, colony morphologies, biofilms in human
infection, patient groups at heightened risk for nosocomial infection.

Laboratory Session 6
Effectiveness of Disinfectants by Disk Diffusion

PURPOSE
1) To test for potential antibiotic production between organisms.
2) To learn the procedures for testing bacterial susceptibility to antimicrobial agents.

BACKGROUND
Last week samples were collected to establish the model of nosocomial infection. Once again,
the cotton swab represented your finger or a device such as a catheter, the Adelphi University
science building hallways represented the hospital, the nutrient agar plate represented the patient
and the bacteria now growing on the nutrient agar represent the nosocomial infection.

The below is adapted from: World Health Organization, Model Formulary, 2004
“A microbiology teaching laboratory does not have the same need for disinfections and sterilization as a
hospital operating room, but you will use many of the same techniques for controlling microbial
contamination in this laboratory assignment. Furthermore, the techniques used in this laboratory exercise
27
are used in other settings, including the food industry, drinking water treatment facilities, and sewage
treatment plants. This industry of killing microbes is one of the leading industries as you can see from the
vast amounts of disinfectants and antiseptics available at your local store.

One way of killing microbes is via heat. Usually a machine called an autoclave is used to kill
all bacteria but in our lab we usually use a live flame to kill off any bacteria on our inoculating
loop. One other way of killing microbes is with the use of chemical agents.

A disinfectant is a chemical agent, which destroys or inhibits growth of pathogenic micro-

organisms in the non-sporing or vegetative state. Disinfectants do not necessarily kill all
organisms but reduce them to a level which does not harm health or the quality of perishable
goods.

Antiseptics are not meant to be used on inanimate objects, such as instruments and surfaces.
Antiseptics are designed to be used for reducing or destroying microorganisms on the skin or
mucous membranes without damaging these tissues. They usually do not have the same killing
power as chemicals used for high-level disinfection of inanimate objects.

One way to test a bacterium's susceptibility to antibiotics, disinfectants or antiseptics is using the
agar diffusion method. This involves the use of disks soaked in a known concentration of
antibiotic, or a disinfectant or antiseptic on a fresh inoculum of bacteria. The bacteria are plated
on nutrient agar medium. The plates with the bacteria and disks are incubated and tested for
growth. A clear zone of no growth around the disk is called the zone of inhibition (Fig. 1 )
(Figure 1 From: microbeonline.com and www.sciencedirect.com) This zone is usually used to
determine the level of effectiveness of the treatment on the bacteria.

Figure 1 From: microbeonline.com and www.sciencedirect.com

MATERIALS

 Two Nutrient Agar Plates

 2 prepared cultures of unknown bacteria from Lab #1
 2 Sterile Swabs
 Four sterile filter paper disks
 2 test tubes sterile water
 Various disinfectant antimicrobial products
 Wax Pencil
28
PROCEDURES

1. Take one plate and use the wax pencil to draw four quadrants on the plastic.
(Fig. 2a)
2. Place the inoculation loop over a Bunsen burner to sterilize.
3. Let the loop cool.
4. Use loop to gather sample of bacterial colony from lab #1 and place the
sample in the center of the agar.
5. Saturate a sterile swab in sterile water (provided at your workstation).
6. Swab evenly over the entire surface of the plate in the following manner:
a. Move the swab back and forth over half of the plate in quadrants 1 and 2
(Fig. 2b)
b. Rotate the plate 90 degrees and swab quadrants 2 and 3 back and forth
(Fig. 2c)
c. Rotate the plate 90 degrees and swab quadrants 3 and 4 back and forth
(Fig. 2d)
d. Rotate the plate 90 degrees and swab quadrants 4 and 1 back and forth
(Fig. 2e)
This will ensure proper growth of bacteria over the entire plate.

Manuscripts for Lab 6 and 7

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5091001/ (hand sanitizer)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6172160/ (pomegrantite)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466868/ (various plants)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4669908/ (drug resistance in

Urinary Tract)

29
 7. Use the four disinfectant products to test and label each quadrant with the
disinfectant product’s name (use abbreviations if possible). Make sure you do
one at a time to avoid confusion.
8. Take a forcep and place into a beaker of ethanol (available at end of lab
bench). Air dry the ethanol off the forcep.
9. Use the forcep to remove one disk of sterile filter paper (available at end of
lab bench) from the dish.
10. Use the forcep to dip the filter paper disk into your chosen disinfectant
(available at end of lab bench).
11. Immediately place the disk in the center of the designated quadrant and tap
with forcep. (once placed do not remove or move because the disinfectant is
immediately absorbed into the agar)
12. Repeat steps 8 through 11 for each of the remaining 3 disinfectant products.
13. Repeat steps 1 through 8 for a second colony sample from lab #1.
14. Invert each plate (disk will not come off) and bring to the front for incubation
at 37 degrees Celsius.
15. Think about natural compounds that you think may have antimicrobial
properties. You can bring samples of these materials to next lab.

RESULTS
Measure Zones Next Week. Record your results from disinfectant test. Record whether there
was growth or zones of inhibition when the disinfectants were used.
30
DISCUSSION ASSIGNMENT QUESTIONS

1. Do you think the disinfectants you used are effective antimicrobials? Why?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

2. Why do microorganisms differ in their response to disinfectants?

NOTES DURING LECTURE/DISCUSSION:

31
 FINAL SUBMITTED ANSWER:

3. Which microorganisms are most susceptible to disinfectants?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

4. What are bacterial endospores?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

5. Why are bacterial endospores a problem in the hospital environment?

NOTES DURING LECTURE/DISCUSSION:

32
 Laboratory Session 7
Antimicrobial Effect of Natural Compounds
PURPOSE
 To test for potential antibiotic activity of natural compounds
 To learn the procedures for testing bacterial susceptibility to antimicrobial agents.
BACKGROUND
Last week samples were collected to establish the model of nosocomial infection. Once again,
the cotton swab represented your finger or a device such as a catheter, the Adelphi University
science building hallways represented the hospital, the nutrient agar plate represented the patient
and the bacteria now growing on the nutrient agar represent the nosocomial infection.

“In 1928, Alexander Fleming observed that when applied, a mold (Penicillium notatum), could
destroy colonies of the bacterium Staphylococcus aureus. Fleming was able to isolate the
antimicrobial principle in this mold which led to the development of medicine that could combat
bacteria inside the body. In nature we may find substances and compounds that appear to be
resistant to bacterial infection or that may inhibit bacterial growth. The discovery and
development of such compounds would be very advantageous to the study and application of
health in humans. “https://www.thoughtco.com/history-of-penicillin-1992304

MATERIALS
 Two Nutrient Agar Plates
 2 cultures from lab #1
 2 Sterile Swabs
 2 test tubes of sterile water
 8 sterile filter paper disks
 Wax Pencil

PROCEDURES
Procedures:
1. Take one plate and use the wax pencil to draw four quadrants on the plastic.
(Fig. 2a)
2. Place the inoculation loop over a Bunsen burner to sterilize.
3. Let the loop cool.
4. Use loop to gather sample of bacterial colony from lab #1 and place the
sample in the center of the agar.
5. Saturate a sterile swab in sterile water (provided at your workstation).
6. Swab evenly over the entire surface of the plate in the following manner:
a. Move the swab back and forth over half of the plate in quadrants 1 and 2
(Fig. 2b)
b. Rotate the plate 90 degrees and swab quadrants 2 and 3 back and forth
(Fig. 2c)
c. Rotate the plate 90 degrees and swab quadrants 3 and 4 back and forth
(Fig. 2d)

33
 d. Rotate the plate 90 degrees and swab quadrants 4 and 1 back and forth
(Fig. 2e)
This will ensure proper growth of bacteria over the entire plate.

7. Label each quadrant with the natural compound of choice (use abbreviations if
possible). Make sure you do one at a time to avoid confusion.
8. Take a forcep and place into a beaker of ethanol (available in front of class).
Air dry the ethanol off the forcep.
9. Use the forcep to remove one disk of sterile filter paper (available in front of
class) from the dish.
10. Use the forcep to dip the filter paper disk into your chosen natural product
(available in front of class).
11. Immediately place the disk in the center of the designated quadrant and tap
with forcep. (once placed do not remove or move because the disinfectant is
immediately absorbed into the agar)
12. Repeat steps 8 through 11 for each of the remaining 3 disinfectant products.
13. Repeat steps 1 through 9 for a second colony sample from lab #1.
14. Invert each plate (disk will not come off) and bring to the front for incubation
at 37 degrees Celsius.

RESULTS

34
Record your results from the test. Record whether there was growth or zones of inhibition when
the natural compounds were used.

DISCUSSION ASSIGNMENT QUESTIONS

https://www.ncbi.nlm.nih.gov/pubmed/32162052 (alganate films for wounds)

https://www.ncbi.nlm.nih.gov/pubmed/31516355 Ehretia Seratta (natural compound)

35
Questions:

1. Why might effective natural antimicrobial compounds be a beneficial development?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

2. Why do you think some natural compounds are able to kill bacteria?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

36
 Laboratory Session 8
Antimicrobial Medicines

PURPOSE
 To learn the procedures for testing bacterial susceptibility to antimicrobial agents.
 To compare the susceptibilities of unknown samples taken from the environment.
BACKGROUND
Last week samples were collected to establish the model of nosocomial infection. Once again,
the cotton swab represented your finger or a device such as a catheter, the Adelphi University
science building hallways represented the hospital, the nutrient agar plate represented the patient
and the bacteria now growing on the nutrient agar represent the nosocomial infection.

The term antibiotic was coined from the word antibiosis, meaning against life. Antibiotics arc substances
that are produced by microorganisms that inhibit the growth of other microorganisms within the body.

Antibiotics are some of the most frequently prescribed medications in use and over one hundred
types of are available to fight bacterial infection. Antibiotics can be taken orally, intravenously
(typically in more serious cases, such as deep-seated systemic infections), or topically (such as
eye drops or ointments).

Today, because of widespread antibiotic use, pathogenic microorganisms have become more
and more resistant to these drugs. We now have many microorganisms that are not sensitive to
these antibiotics. It was once believed that antibiotics will always be available to fight infections
but in this era there are so many mutations in microorganisms that this has become a widespread
research topic.

Antibiotic resistance occurs when resistant strains of bacteria are left to grow and multiply after
treatment with an antibiotic (this is frequently due to the patients' repeated misuse of
antibiotics). Antibiotic resistance is becoming an increasingly serious problem, as common
infections may eventually become untreatable with known antibiotics.

One way to test bacterium's susceptibility to antibiotics is using the agar diffusion method. This
involves the use of commercially produced antibiotic impregnated disks of known concentration
on a fresh inoculum of bacteria. The bacteria are plated on standard agar medium called
Mueller-Hinton (MH) agar. MH agar is a specially designed agar with specific nutrients, a pH
of 7.4, and cation concentrations that allow the diffusion of antibiotics. The plates with the
bacteria and disks are incubated and tested for growth. A clear zone of no growth around the
disk is called the zone of inhibition. This zone is usually used to determine the level of
effectiveness of the antibiotic on the bacteria

MATERIALS
 Two Mueller Hinton Plates
 2 cultures from Lab #1
 2 Sterile Swabs
 8 various antibiotic impregnated disks
37
  Wax Pencil

PROCEDURES
1. Take one Mueller Hinton plate and use the wax pencil to draw four quadrants
on the plastic. (Fig. 2a)
2. Place the inoculation loop over a Bunsen burner to sterilize.
3. Let the loop cool.
4. Use loop to gather sample of bacterial colony from lab #1 and place the
sample in the center of the agar.
5. Saturate a sterile swab in sterile water (provided at your workstation).
6. Swab evenly over the entire surface of the MH plate in the following manner:
a. Move the swab back and forth over half of the plate in quadrants 1 and 2
(Fig. 2b)
b. Rotate the plate 90 degrees and swab quadrants 2 and 3 back and forth
(Fig. 2c)
c. Rotate the plate 90 degrees and swab quadrants 3 and 4 back and forth
(Fig. 2d)
d. Rotate the plate 90 degrees and swab quadrants 4 and 1 back and forth
(Fig. 2e)
This will ensure proper growth of bacteria over the entire plate.

7. Label each quadrant with the name of the antibiotic (use abbreviations if
possible).
Discussion
38
 Manuscripts for Lab 8 and antibiotic plate picture on BSL1 mixture
Website to find medical publications: www.pubmed.gov

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525210/ (natural product in combination with

antibiotics)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072311/ (zone of inhibition against MRSA)

39
 Questions:

1. Define an antimicrobial agent.

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

2. What is meant by antimicrobial resistance? Susceptibility?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

3. Why are pure cultures used for antimicrobial susceptibility testing?

NOTES DURING LECTURE/DISCUSSION:

40
FINAL SUBMITTED ANSWER:

4. Would it be acceptable to use a mixed culture for this test? Why?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

5. Describe a mechanism of bacterial resistance to antimicrobial agents.

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

41
6. Could an organism that is susceptible to an antimicrobial agent in laboratory testing
fail to respond to it when that drug is used to treat the patient? Explain.

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

7. Are antibacterial agents useful in viral infections? Explain.

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

42
43
 Laboratory Session 9
Lab Exam 2
PURPOSE
To test students on drugs and conditions that leave patients at a higher risk for infection, atrophy
of the immune system, the nature of disinfectant, antiseptics and antibiotics and the difference
between disinfection and sterilization.

BACKGROUND
None
MATERIALS
None
PROCEDURES
None
RESULTS
None
DISCUSSION QUESTIONS
None

Laboratory Session 10
Simple Stains
PURPOSE
 To learn the value of simple stains in studying basic microbial morphology
 To examine the basic bacterial morphology of four (4) environmental samples
BACKGROUND
Wet mounts of bacterial cultures can be very informative, but they have limitations.
Bacteria bounce about in fluid suspensions with Brownian movement or true motility, and are
difficult to visualize sharply. We can see their shapes and observe their activity under a cover
glass, but it is difficult to form a complete idea of their morphology.
An important part of the problem is the minute size of bacteria. Because they are so
small and have so little substance, they tend to be transparent, even when magnified in subdued
light. The trick, then, is to find ways to stop their motion and tag their structures with something
that will make them more visible to the human eye. Many sophisticated ways of doing this are
known, but the simplest is to smear out a bacterial suspension on a glass slide, "fix" the
organisms to the slide, then stain them with a visible dye (Koch and his coworkers first thought
of this more than 100 years ago).
The best bacterial stains are aniline dyes (synthetic organic dyes made from coaltar
products). When they are used directly on fixed bacterial smears, the contours of bacterial
bodies are clearly seen. These dyes react in either an acidic, basic, or neutral manner. Acidic or
basic stains are used primarily in bacteriologic work. The free ions of acidic dyes are anions
(negatively charged) that combine with cations of a base in the stained cell to form a salt. Basic

44
dyes possess cations (positively charged) that combine with an acid in the stained material to
form a salt. Bacterial cells are rich in ribonucleic acid (contained in their abundant ribosomes)
and therefore stain very well with basic dyes. Neutral stains are made by combining acidic and
basic dyes. They are most useful for staining complex cells of higher forms because they permit
differentiation of interior structures, some of which are basic, some acidic. Cells and structures
that stain with basic dyes are said to be basophilic. Those that stain with acid dyes are termed
acidophilic. Stained bacteria can be measured for size and are classified by their shapes and
groupings.
Bacteria are so small that their size is most conveniently expressed in micrometers
(symbol/-Lm). A micrometer is a thousandth part of a millimeter, and 1/1 0,000 of a centimeter,
or 1125,400 of an inch. Bacteria vary in length and diameter, the smallest being about 0.5 to 1 /-
Lm long and approximately 0.5 /-Lm in diameter, whereas the largest filamentous forms may be
as long as 100 /-Lm. Most of those you will see in this course are at the small end of the scale,
measuring about 1 to 3 I-Lm in length. Small as they are in reality, their images should loom
large in your mind as the agents of infection in patients for whom you will be caring.
Bacteria have rigid cell walls and maintain a constant shape. Therefore, they can be classified
on the basis of their form. Bacteria have three basic shapes: spherical (round), rod shaped, or
spiraled. A round bacterium is called a coccus (plural, cocci). A rod-shaped organism is called
a bacillus (plural, bacilli) or simply a rod. A spiraled bacterium with at least two or three
curves in its body is called a spirillum (plural, spirilla). Long sinuous organisms with many
loose or tight coils arc called spirochetes. The patterns formed by bacterial cells grouping
together as they multiply are often characteristic for individual bacterial genera or species.
Cocci may occur in pairs (diplococci), chains (streptococci), clusters (staphylococci), or
packets of four (tetrads), and are seldom found singly.
Rod-shaped bacteria (bacilli) generally occur as individual cells, but they may appear as
end-to-end pairs (diplobacilli) or line up in chains (streptobacilli). Some species tend to
palisade, that is, line up in bundles of parallel bacilli, others may form V, X, or Y figures as they
divide and split. Some may show great variation in their size and length (pleomorphism).
Spiraled bacteria occur singly and usually do not form group patterns.
MATERIALS
 2 bacterial cultures from lab #1
 Methylene Blue Stain
 Staining tray
 4 glass slides
 Staining rack
 Distilled water
 Gloves
 Bunsen burner
 Wax pencils
 Inoculating loop
 Bilbulous paper
 Microscope

PROCEDURES
1. Start with a clean slide.
45
 2. Draw a small circle in the center of the slide with a wax pencil.
3. Label one end of the slide with your initials and the location where you obtained your
sample.
4. Turn the slides over so that the unmarked side is up. (When slides are to be stained,
pen or pencil markings should always be placed on the underside so that the mark
will not smear, wash off, or run into the smear itself.)
5. With your inoculating loop, place a loopful of water in the ringed area of the slide.
6. Using proper aseptic transfer techniques mix a small amount of bacteria in the water
and spread it out. Be certain the smear is only lightly turbid. Repeat this step until
smears of all slides have been made.
7. Allow the smears to air dry. You should be able to see a thin white film on each
slide. If not, add another loopful of water and more bacteria as in step 4.
8. Heat-fix the smears by passing the slides rapidly through the Bunsen flame three
times so that the smears will not wash off.
9. Place the slides on a staining rack and flood them with methylene blue. Leave the
stain on for three minutes.
10. Wash each slide gently with distilled water, drain off excess water, blot (do not rub)
with bilbulous paper, and let the slides dry completely in air.
11. Examine all slides with three microscope objectives (ultimately at 1000X).
Record your results in the table and draw what you see.

RESULTS

Location of Stain Color Coccus, Rod Cell

Sample or Spiral Groupings

46
47
48
DISCUSSION ASSIGNMENT QUESTIONS

1. Define acidic and basic dyes. What is the purpose of each?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

2. What is the purpose of fixing a slide that is to be stained?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

3. Why are specimens to be stained suspended in sterile saline or distilled water?

49
 NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

4. Which of the microscope objectives is most satisfactory for studying bacteria? Why?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

5. How does a stained preparation compare with a hanging drop for studying the
morphology and motility of bacteria?

NOTES DURING LECTURE/DISCUSSION:

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 FINAL SUBMITTED ANSWER:

6. List and define the basic shapes of bacteria. What are the dimensions of an average
bacillus in micrometers? In centimeters?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

7. List at least three types of bacteria whose names reflect their shapes and arrangements,
and state the meaning of each name.

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

51
8. For what reason do we need to stain bacteria?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

52
 Laboratory Session 11 +12
Gram Stain
PURPOSE
 To learn the Gram-stain technique and to understand its value in the study of
bacterial morphology
 To observe basic bacterial morphology
BACKGROUND
The simple staining procedure makes it possible to see bacteria clearly, but it does not
distinguish between organisms of similar morphology.
In 1884, a Danish pathologist, Christian Gram, discovered a method of staining bacteria
with pararosaniline dyes. Using two dyes in sequence, each of a different color, he found that
bacteria fall into two groups. The first group retains the color of the primary dye: crystal violet
(these are called gram positive). The second group loses the first dye when washed in a
decolorizing solution but then takes on the color of the second dye, a counterstain, such as
safranin or carbol fuchsin (these are called gram negative). An iodine solution is used as a
mordant (a chemical that fixes a dye in or on a substance by combining with the dye to form an
insoluble compound) for the first stain.
The exact mechanism of action of this staining technique is not clearly understood.
However, it is known that differences in the biochemical composition of bacterial cell walls
parallel differences in their Gram-stain reactions. Gram-positive bacterial walls are rich in
tightly linked peptidoglycans (protein-sugar complexes) that enable cells to resist
decolorization. Gram-negative bacterial walls have a high concentration of lipids (fats) that
dissolve in the decolorizer (alcohol, acetone, or a mixture of these) and are washed away
with the crystal violet. The decolorizer thus prepares gram-negative organisms for the
counterstain.
The Gram stain is one of the most useful tools in the microbiology laboratory and is
used universally. In the diagnostic laboratory, it is used not only to study microorganisms in
cultures, but it is also applied to smears made directly from clinical specimens. Direct, Gram-
stained smears are read promptly to determine the relative numbers and morphology of bacteria
in the specimen. This information is valuable to the physician in planning the patient's treatment
before culture results are available. It is also valuable to microbiologists, who can plan their
culture procedures based on their knowledge of the bacterial forms they have seen in the
specimen.
The numerous modifications of Gram's original method are based on the concentration
of the dyes, length of staining time for each dye, and composition of the decolorizer. Hucker's
modification, to be followed in this exercise, is commonly used today. The choice of
decolorizing agent depends on the speed wanted to accomplish this step. The slowest agent,
95% ethyl alcohol, is used in this exercise to permit the student to gain experience with
decolorization. Acetone is the fastest decolorizer, while an equal mixture of 95% ethyl alcohol
and acetone acts with intermediate speed. The acetone-alcohol combination is probably the
most popular in diagnostic laboratories.
Although you are not directed to do so in this exercise, to properly judge the adequacy of
a stained preparation, smears of a known gram-positive and gram-negative organism arc placed

53
on the same slide as the 1.111known organism or patient specimen. These two smears serve as
the controls, because on a well-stained smear, the appropriate Gram reaction (Gram positive or
Gram negative) of these control organisms should be seen. If these organisms do not show their
correct type of staining, the Gram reaction of the unknown is not reliable, and the staining
procedure must be repeated.
MATERIALS
 Known bacterial cultures from lab #1
 Crystal Violet Stain
 Gram’s Iodine
 Ethyl Alcohol, 95%
 Safranin (slightly toxic)
 Staining tray
 4 glass slides
 Staining rack
 Distilled water
 Gloves
 Bunsen burner
 Wax pencils
 Inoculating loop
 Bilbulous paper
 Microscope

PROCEDURES
1. Start with a clean slide.
2. Draw a small circle in the center of the slide with a wax pencil.
3. Label one end of the slide with your initials and the location where you obtained your
sample.
4. Turn the slides over so that the unmarked side is up. (When slides are to be stained,
pen or pencil markings should always be placed on the underside so that the mark
will not smear, wash off, or run into the smear itself.)
5. With your inoculating loop, place a loopful of water in the ringed area of the slide.
Using proper aseptic transfer techniques mix a small amount of bacteria from lab #1
in the water and spread it out. Be certain the smear is only lightly turbid. Repeat this
step until smears of all slides have been made.
6. Allow the smears to dry. You should be able to see a thin white film on each slide. If
not, add another loopful of water and more bacteria as in step 4.
7. Heat-fix the smears by passing the slides rapidly through the Bunsen flame three
times so that the smears will not wash off.
8. Stain each smear by the following procedures (this is Hucker’s modification of the
Gram stain):
9. Flood slide with crystal violet. Allow to stand for five minutes.
10. Wash off with tap water.
11. Flood with Gram’s iodine (a mordant). Leave for two minutes.
12. Wash off with tap water.

54
 13. Decolorize with alcohol (95%) drop-wise until no more color washes off (10
seconds!). This is a most critical step. Be careful not to over-decolorize, as many
gram-positive organisms may lose the violet stain area easily and this appear to be
gram negative after they are counterstained.
14. Wash off with tap water.
15. Apply Safranin (the counterstain) for two minutes.
16. Wash off with tap water.
17. Drain and blot gently with bulbulous paper. Air dry the slide thoroughly before you
examine the preparation under the microscope.
18. Repeat steps 1-17 with three (3) additional environmental samples obtained from lab
#1.
19. Examine all slides under oil with the oil-immersion objective (1000X).
20. Record your observations in table under results.

RESULTS

Results:

Location where Color Gram Stain Bacteria Shape

sample was (Purple or Reaction (+) or (Bacillus,
obtained Pink) (-) Coccus,
Spirillus)

DISCUSSION QUESTIONS

55
Questions:

1. What is the function of the iodine solution in the Gram stain? If it were omitted, how
would staining results be affected?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

2. What is the purpose of the alcohol solution in the Gram stain?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

56
3. What counter-stain is used? Why is it necessary? Could colors other than red be used?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

4. What is the advantage of the Gram stain over the simple stain?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

57
5. In what kind of clinical situation would a direct smear report from the laboratory be or
urgent importance?

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

6. Describe at least two conditions in which an organism might stain Gram variable.

NOTES DURING LECTURE/DISCUSSION:

FINAL SUBMITTED ANSWER:

58
 Laboratory Session 12 - 14
Identification of unknowns using Gram Staining and Cell
Morphology

PURPOSE
1) To use the knowledge and laboratory skill learned throughout the semester to identify
unknowns. Each bench will be provided with colonies of the 5 known bacteria:
-Bacilus subtilus
-Clostridium sporenges
-E. Coli
-Seratia marcescens
-Staphylococcus epidermidis
2) To use the known colony features, cell morphology and gram staining properties to identify
the above five bacteria

BACKGROUND
MATERIALS
Solid Agar Cultures with individual colonies of
-Bacilus subtilus
-Clostridium sporenges
-E. Coli
-Seratia marcescens
-Staphylococcus epidermidis

PROCEDURES
Use colony morphology observations and also use gram staining to determine cell shape and cell
wall make-up.
RESULTS
1___ __ _
2___ __ __
3____ _ __
4____ ____
5____ ____
DISCUSSION ASSIGNMENT QUESTIONS
None
Laboratory Manual Due at end of session

59
“STUDENT AGREEMENT ON LABORATORY SAFETY
(University Copy)

I have read the Laboratory Safety Statement of the Department of Biological Sciences, Adelphi
University, for course 0105-208-Laboratory Sections and I understand its content. I agree to
abide by all laboratory rules set forth by the instructor and stated in the laboratory manual. I
understand that my safety is entirely my own responsibility and that I may be putting myself and
others in danger if I do not abide by all the rules set forth by the instructor.

NAME OF STUDENT (PRINT): ________________________

SIGNATURE OF STUDENT: ________________________

DATE: ________________________”

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