Intro to Microbiology
Mrs. Cox
(Adapted from Techniques in
Microbiology, A Student Handbook
by John M. Lammert, 2007)
2014-15
Safety First!
• Wash your hands before you begin and after you
complete your work. Also, before you leave the
lab for any reason.
• Keep your personal items (coat, cosmetics,
backpack, electronic devices) away from the lab
bench.
• Dress appropriately for lab: closed-toed shoes,
safety glasses, tie back long hair, wear gloves,
and apron.
• Absolutely no food or beverages (even water) in
the lab while working with bacteria.
• Keep hands away from your mouth and eyes.
• Do not remove cultures, reagents or other
materials from the lab without permission.
More Safety and Getting Started
• Decontaminate your work surface with a disinfectant
before you begin and after you complete your work.
• When using a bunsen burner, position it so that you
will not burn yourself, and turn off the gas when the
flame is not needed.
• Keep bacteria cultures in a test tube rack or other
holder when working at the bench or walking around
the lab.
• Place all contaminated tubes, plates and waste
materials in appropriate receptacles for subsequent
sterilization.
• If anything containing living bacteria spills, cover
with a paper towel and disinfectant. Allow to remain
on the spill for at least 20 minutes!
This is what we are trying to
avoid: CONTAMINATION!
http://runforsushi.blogspot.com/2011/05/contamination-station.html
What is the difference between
disinfection and sterilization?
• Sterilization is the process in which all living
cells, spores, and viruses are completely
destroyed or removed from an object or
environment.
• Disinfection is the killing, inhibition, or
removal of microorganisms that cause
disease. Disinfection may not necessarily
eliminate spores or all of the microorganisms
from an object or environment.
From: http://microbiology.mtsinai.on.ca/faq/cleaning.shtml
The Autoclave
• The autoclave is essentially a high temperature
pressure cooker!
• It creates moist heat at temperatures high enough
to kill bacterial endospores.
• Steam under pressure creates the necessary
121oC.
• The sterilization time for biohazard waste is at
least 45 minutes.
• Materials sterilized in an autoclave must be able to
withstand the high temperatures without breaking
down or melting.
• Plastic petri dishes are sterilized in autoclaveable
plastic bags.
Autoclave Safety
• Do not operate the autoclave without
supervision.
• Open the door slowly, only an inch at the
end of a sterilization cycle.
• Wait 10 minutes before removing objects
from an autoclave. Super heated water
could boil after autoclaving if it is even
slightly agitated.
• Wear insulated gloves when unloading hot
materials.
Adjusting the Bunsen Burner
• We will use a Bunsen burner to sterilize
instruments used at the lab bench, like the
inoculating loop, the bacteria spreader,
and the mouth of broth tubes.
• We will light the Bunsen
burner with a striker.
• The hottest point is at the
tip of the bright blue cone.
• We adjust the size of the
flame by opening or
closing the air holes.
http://www.uwplatt.edu/chemep/chem/chemscape/labdocs/catofp/bunsbur/bunsbur2.htm
Bacteriology…
Bacteria are ALIVE!
Bacteria are alive! That means they…
• REPRODUCE (quickly!)
• Maintain HOMEOSTASIS (respiration and excrete
waste)
• Contain GENETIC MATERIAL
• Use RAW MATERIALS for energy
• CHANGE over time
• Are made of CELLS
• GROW and DEVELOP
• RESPOND to their environment
So…you need to know your bacteria’s ideal
growing condidtions:
• Is it AEROBIC, ANAEROBIC or FACULTATIVE?
• What does FACULTATIVE mean?
• We do not have the capability to grow ANAEROBIC
bacteria this year…do you need to make a change?
• What temperature does it thrive in? What are the upper
and lower temperature limits?
• We have a 37oC incubator. Room temperature is also
an option (25oC). That is all.
• We should probably begin recording daily
temperatures of our incubator to ensure it’s not
getting too hot or too cold!
What kind of media (food) does your bacteria
prefer?
• Most will be good with basic Nutrient Agar or Broth. It
contains all the necessary “food” for most bacteria.
• Other types of media are either SELECTIVE or
DIFFERENTIAL or both.
• Selective means the media contains an ingredient
that selects FOR a certain type of bacteria:
MacConkey Agar contains ingredients selects FOR
Gram Negative organisms.
• Differential means the media contains an ingredient
that differentiates between two different
characteristics of bacteria:
• MacConkey Agar contains lactose which
distinguishes between lactose fermenters and non
lactose fermenters
The Gram Stain…
• Is the first, most basic step in identifying your bacteria.
• It allows you to determine the bacteria’s shape (rod,
cocci, etc.)
• It also breaks all bacteria into two large groups: Gram
positive and Gram negative.
• The gram stain is a multistep process that uses several
different stains: Crystal Violet, Gram’s iodine, and
Safranin.
• These stains may or may not be absorbed by the
bacteria cell wall, giving the bacteria a distinct purple or
red color.
Gram Staining…
• Gram POSITIVE bacteria will be PURPLE in color.
• Gram NEGATIVE bacteria will be pink-ish RED in color.
Some bacteria contain lots of PEPTIDOGLYCAN and
others don’t. If they have lots of PEPTIDOGLYCAN,
they will be GRAM POSITIVE.
Homework
Homework: Know your bacteria name, and be sure
to research its gram stain characteristics, the
optimal growth conditions for your organism and
where it is commonly found in nature. You need
to know why you chose this bacteria and how it
connects to your project.
Supplies you will need for bacteriology…
• Your bactierial culture (order 2!)
• Pre-poured agar plates…lots of them…probably 5
sleeves of 25-30!
• Gloves
• Micropipettor tips (of various sizes)
• Nutrient broth (pre-made is easiest!)
• What else? Maybe sterile, blank antibiotic discs, maybe
known antibiotic discs (for comparison)
• A sharpie