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9/4/2014

Lab 1: Introduction to Microbiology


Lab, Microscopy and the Ubiquity of
Microorganisms
Fall 2014

What is Microbiology?
Microbiology the study of organisms that are difficult
or impossible to observe unaided.

Bacteriology
(Bacteria)

Virology
(Viruses)

Mycology
(Fungi)

Medical Microbiology
Disease causing microbes.

Environmental Microbiology
Studies microorganisms outside of medical importance.

What are Microorganisms?


Microorganisms are single-celled, clustered or
multicellular organisms.
These are microscopic organisms that are to small
to be seen with the naked eye.
They have a great deal of diversity and are
important in many ecosystem processes.

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Germ Theory
Microorganisms are the causative agents for
many diseases and the spread of disease goes
hand in hand with their transmission.
Kochs Four Postulates:
Step 1:
Association

Step 2:
Isolation

Step 3:
Inoculation

Step 4:
ReIsolation

Important Terms
Nosocomial An infection not present upon admission
to a hospital but incurred while being treated there.
Aseptic Free of pathogenic organisms.
Pathogen Disease causing agent.
Inoculation Implantation of micro-organism into or
upon culture media.
Turbid Cloudy appearance of a nutrient solution in a
test tube due to growth of microbe population.

Safety in Lab

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Disposal of Materials
Sterilization /decontamination for most
materials prior to use or being disposed off.
Sterilization techniques commonly used:
Autoclave
Incineration
UV cross-link

Culture tubes: Can be washed and reused after


autoclaving.
Plastic petri-plates, disposable needles &
swabs: Melts during autoclaving and disposed
of after autoclaving.

Containment
Containment of Micro-organisms:
Primary Containment Protection of
the Lab personnel and the lab
environment.
Secondary Containment Protection
of the outside Lab environment from
exposure to infectious organisms.

Always Follow The Rules For The


Safety of Yourself and Others!

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General Lab Safety

No open toed shoes, sandals or flip flops.


No short pants or skirts.
No food or drinks!!!!!!!!
Baggy sleeves should be avoided as they can catch fire. Wear a lab coat.
Become knowledgeable about safety gear in lab.
Locate eyewash, fire extinguisher, fire blanket and first-aid kit.
Wash hands before and after lab (even if gloves were worn).
Tie back hair (fire and safety hazard).
Disinfect lab bench before and after lab.
Books, bags and other items should not be kept on work bench.
The open flames of a Bunsen burner is a fire hazard (use common sense).
Skin and eyes are common portals of entry for microorganisms.
Do not touch face in lab (do not apply make up or contact lens either!).

Bio-Safety Level (BSL)


BSL 1-4 based on their ease of transmission and
pathogenicity: Each level has a set of minimum standards
and with regard to laboratory practices , equipment and
facilities.

BSL 1: generally do not cause disease in healthy person and


require very few specialized techniques.
Most organisms in General Microbiology lab

BSL 4: easily transmitted and cause life-threatening disease


Requires full-body spacesuits, respirators, showers, etc.
Ex. Ebola virus and smallpox

An introduction to Microscopy
Antonie Philips van Leeuwenhoek:
(October 24, 1632 August 26, 1723)
commonly referred to as "the Father
of Microbiology", and considered to
be the first microbiologist.
Bright field or light microscope
because objects examined appear as
dark objects in bright visual field.

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Parts of a Microscope

Magnification and Resolution


Magnification = number of times an object is enlarged.
Total Magnification = Magnification of objective lens times the
magnification of ocular lens.
Ocular Lens are 10x magnification. Objective lenses are 4x, 10x, 40x
and 100x magnification. To figure out the total magnification,
multiple the ocular lens by the objective lens.

Example: (10x ocular)(40x objective) = 400x total magnification


Resolution = the ability to discern points.

0.61

Resolution : =

R = Resolving power of the optical system


= wavelength of the light used
NA = numerical aperture of the object being used

Oil Immersion
Highest
magnification of a
light microscope.
Oil has same
refractive index of
glass and prevents
light loss.

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Microscope Care
Transport Always use both hands. One hand to grasp the
microscope around the arm and the second to support it at the
bottom.
Electric cord Never leave the cord dangling, wrap it loosely
around the base of the scope.
Protection against dust and chemicals If a dust cover is provided,
be sure to use it.
Lens cleaning unacceptable images mostly result from dirty
lenses. Cleaning dirt , dust and stain off the lens is must after each
use. To avoid scratches , only LENS PAPER should be used. Small
amount of alcohol or xylene can be used to remove oily residue.

Common Bacterial Cell Morphologies

Bacillus

Spirillum

Cocci

Todays Lab Work


Environmental Microbe Isolation:
Using culture plates.
Testing different environments.

Microscope use.
Staining of microorganisms and use of
microscopes.

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Isolation of Microbes
This exercise will be for a lab report
Ubiquity of Microbes:
Microbes inhabit almost every environment (ubiquitous).
Although microbes inhabit many locations, their detection requires that they
be:
removed
transferred to growth media

Purpose of this lab : Isolate microbes from the environment


Compare two environments microbial communities
Select 2 environmental sites (ex. sink, door knob, toilet seat, bench station,
water fountain, under the shoe etc).
Transfer bacteria from the environment onto a nutrient agar plate (solid
media)
Using a sterile swab moistened with water inoculate of nutrient agar plate with one
environment
Use another swab for the second with the second environment

Review: How to Use a Microscope


Turn the revolving nosepiece so that the lowest power objective lens (4x
smallest) is clicked into place.
Place the microscope slide onto the stage and fasten it down with the
stage clips.
Look at the objective lens and the stage and turn the focus knob so that
the stage moves up. Move the stage up (slowly) as far as it will go without
letting the objective touch the coverslip.

Never let the objective lens touch the slide!


Now look through the eyepiece and move the focus adjustment knob until
the image comes into focus.
Adjust the condenser for the greatest amount of light.

Review: How to Use a Microscope


Move the specimen slide around using the stage adjustment knob
until the microorganism is in center of your field of view.
Use the fine focus knob to place the microorganism into focus and
re-adjust the condenser for the clearest image.
When you have a clear image of your microorganism with the
lowest power objective, you can now change to the next objective
lenses (10x). You might need to re-adjust the microorganism into
focus and/or re-adjust the condenser for clarity.
When you have finished, lower the stage, click the low power lens
(4x) back into position, remove the slide, unplug your microscope,
wrap the cord loosely around the base and put away into the
proper cabinet.

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Staining Microbes
Most microorganisms are transparent on slides
and nearly impossible to differentiate.
Stains are used for visualization and come in
many varieties:
Simple
Differential
Negative
Positive

For staining microbes


This will not be for the report
You will be using two simples stains with a
partner
One partner will:
Perform a negative stain on E.coli
This will use the dye nigrosin

The other partner will:


Perform a positive stain on E.coli
This will use the dye methyl red or methyl blue

Observe both under oil immersion.

Negative Stain

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Heat Fixing Bacteria

http://loudoun.nvcc.edu/vetonline/VET133/fe
cal%20smear.htm

Positive Simple Stain

Questions to think about


What do the organisms look like at each
magnification?
How do different stains compare to one
another?
When would there be utility to use these
different methods?

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For Next Week


Finish ubiquity of microorganisms exercise.
Aseptic technique (Exercises 1-4, 1-5, 1-6).
Gram Staining (Exercise 3-6).

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