Microbiology Lab Practical
Hazardous Material Information System
Color type of hazard
Number 1 – 4
of the hazard
“4” most hazardous
National Fire Protection Agency
Material Safety Data Sheet
Physical and chemical characteristics
Primary Routes of entry
General precautions for safe handling and use
Emergency and first aid procedures
Personal Protective Equipment
refers to protective clothing, helmets, goggles, or other garments or equipment designed to protect the wearer's body from injury.
Hazard Communication Standard (HCS)
requires all chemicals in the workplace be labeled in a manner
warns of any hazards the chemical may present
Good Laboratory Practice Regulations
Know COMMON SENSE!
prevents unwanted environmental microorganisms from entering a culture.
is a method that prevents the introduction of unwanted
organisms into a culture or an environment. When changing wound dressings, this is used to prevent possible infection. When working with microbial cultures, this is used to prevent introducing additional organisms
into the culture.
AKA Aseptic technique
Chemicals applied to kill microorganisms on surfaces
preparations of chemicals that are meant to be applied to the skin or other living tissue.
any process that eliminates (removes) or kills all forms of microbial life, including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) present on a surface, contained in a fluid, in medication, or in a compound such as biological culture media
Media that permits growth of certain groups of microorganisms while inhibiting the growth of other microorganisms by the addition of an inhibitory substance.
Media that distinguishes between species of bacteria by incorporating chemicals to produce specific kind of growth or change in the medium
Blood and other special nutrients may be added to general purpose media to encourage the growth of fastidious microbes. These specially forfited media are called as enriched media. e.g. Blood agar, Chocolate agar.
Media used to isolate a bacterium by enhancing growth due to added nutrients. This media is designed for the growth of fastidious bacteria.
the smear is stained with a solution of a single dye which stains all cells the same color.
distinguishes two kinds of organisms. An example is the Gram stain technique.
The shape, color, and general appearance of an individual colony of bacteria on a plate
often used to identify the species present
Gram stain morphology
Method of differentiating bacterial shapes into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls.
The rupturing of red blood cells
Polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of bacteria (but not forming the cell wall)
Requires oxygen to grow
Can use oxygen, but also has anaerobic methods of energy production
A commonly-used bacterial indicator of sanitary quality of foods and water
A Gram negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms
A facultative anaerobic, Gram-positive coccus and is the most common cause of staph infections
Loss of electrons or an increase in oxidation state
Process of deriving energy from the oxidation of organic compounds
A disaccharide sugar that is formed from galactose and glucose
An enzyme that breaks starch down into sugar
The removal of an amine group from a molecule
A chemical reaction which releases carbon dioxide
An enzyme that functions to catalyze the decomposition of hydrogen peroxide to water and oxygen
Enzyme that functions to catalyze an oxidation-reduction reaction involving molecular oxygen as the electron acceptor.
Enzyme that catalyzes the hydrolysis of urea into carbon dioxide and ammonia
Bacterium partially hemolyses RBC’s with a partial clearing zone around bacterial growth; sometimes with greenish pigment caused by reduction of hemoglobin.
Complete lysis of RBC’s with total clearing zone around bacterial growth.
No lysis of RBC’s with no clearing zone around the bacterial growth.
Examples of obligate anaerobes
Examples of facultative anaerobes
Blood Agar Plate
Enriched with blood to enhance the growth of fastidious bacteria usually pathogenic. Groups of bacteria can be determined by hemolysis of red blood cells by bacterium.
Mannitol Salt Agar
Selects and differentiates among members of genus Staphylococcus.
Mannitol Salt Agar is selective for...
Selective for gram positive staphylococci because staph is salt tolerant and ferments mannitol sugar.
Acidification indicated by...
phenol red pH indicator which turns from red to yellow
What do pathogenic staphylococci form on mannitol salt agar?
small yellow colonies surrounded by yellow zones.
Eosin Methylene Blue Agar
Selects and differentiates members of Enterobacteriaciae. Differentiates genera of coliform group from other genera of Enterobacteriaciae on the basis of lactose fermentation.
EMB Plate inhibits the growth of...
gram positive bacteria
What is the basis of classification of bacteria according to the gram stain?
the cell wall
Gram Positive Bacteria
have a thick layer of peptidoglycan external to the cytoplasmic membrane. more highly cross-linked peptidoglycan structure
contains Lipoteichoic acids (LTA)
Gram Negative Bacteria
have a thin layer of peptidoglycan located between the cytoplasmic membrane and a second membrane called the outer membrane. This region is known as the periplasmic space.
contain Lipopolysaccharides (LPS)
Primary Stain in Gram Stain
how long do you let stand?
Crystal Violet Solution
30-seconds to a minute
Mordant in Gram Stain
how long do you let stand?
30-seconds to a minute
Decolorizer in Gram Stain
how long do you let stand?
20% acetone :80% ethyl alcohol
Counter Stain in Gram Stain
how long do you let stand?
30-seconds to a minute
Steps to making a heat fixed smear...
Using a sterilized inoculating loop place one drop of water on a clean glass microscope slide
Using a sterilized inoculating loop place small amount of bacteria on the clean glass microscope slide next to the drop of water.
With the inoculating loop emulsify the bacteria in the drop of water placed on the clean glass microscope slide.
Sterilize the inoculating loop after use.
Dry the smear by putting it on the slide warmer or let the smear air dry.
Heat fix the smear by putting it on the slide warmer for one minute. Be careful not to overheat the slide.
Gram positive reaction
purple in color
Gram negative reaction
red in color
MacConkey Agar Results pink
(Lactose fermentation colony)
MacConkey Agar Results transparent, clear, or colorless
(Non-lactose fermentation colony)
Selective and differentiates members of Enterobacteriaciae.
Media contains bile salts and crystal violet that inhibits growth of gram positive bacteria
Bubbles = positive
No bubbles = negative
Purple = positive
Light pink or absence of color = negative
Mostly, gram-negative diplococci, gram-negative spiral curved rods
Salmonella, Yersinia pestis, E. coli
when the antibiotic kills the bacteria
when the antibiotic does not kill the bacteria
Most common bacteria causing wound infections is...
where you look through to see the image of your specimen.
Types of eyepieces
monocular & binocular
the long tube that holds the eyepiece and connects it to the objectives.
the rotating part of the microscope at the bottom of the body tube; it holds the objectives.
scanning, low, medium, high, oil immersion) the microscope may have 2, 3 or more objectives attached to the nosepiece; they vary in length (the shortest is the lowest power or magnification; the longest is the highest power or magnification).
part of the microscope that you carry the microscope with.
How to carry a microscope...
one hand to hold the arm and the other hand to support the base
Coarse Adjustment Knob
large, round knob on the side of the microscope used for focusing the specimen; it may move either the stage or the upper part of the microscope.
Fine Adjustment Knob
small round knob within the larger course adjustment knob or separate small round knob on the side of the microscope used to fine-tune the focus of your specimen after using the coarse adjustment knob.
large, flat area under the objectives; it has a hole in it (see aperture) that allows light through; the specimen/slide is placed on the stage for viewing.
shiny, clips on top of the stage which hold the slide in place.
the hole in the stage that allows light through for better viewing of the specimen.
controls the amount of light going through the aperture
Light or Mirror
source of light usually found near the base of the microscope; the light source makes the specimen easier to see
supports the microscope
Magnification of Eyepiece
Magnification of Scanning lens (red)
Magnification of Low power lens (yellow)
Magnification of High power lens (blue)
Magnification of Oil Immersion lens
eyepiece magnification (10x) times objective lens
composed of only one species of microorganism.
there are two or more organisms that have distinct characteristics and can be separated easily.
When unwanted organisms are introduced into the culture
Purpose of Quadrant Streaking
to produce isolated colonies of a microorganism on an agar plate.
AKA streaking for isolation
General Purpose Media
Media that provides enough nutrients in which most any microorganism will utilize for growth. This type of media supports the growth of a wide variety of microorganisms.
Nutrient Agar (NA)
general purpose medium used for the isolation of wide variety of bacteria
Why do we stain?
to increase the contrast
Unstained bacteria appear virtually transparent in brightfield illumination
The most common form of microscopy used in clinical microbiology is...
How long can a urine specimen sit before it is plated? What can be done if it cannot be plated within this time period?
No longer than 2 hours! or bacterial count will not be valid
May be refrigerated up to 24 hours without being plated
What bacteria make up the normal urethral flora?
Females: diphtheroids, lactobacilli, coagulase-negative staphylocci, alpha streptococci and low numbers of Enterobacteriaceae.
Males: diphtheroids, coagulase-negative staphylococci, and Enterococcus species.
What are the most common organisms causing urinary tract infections?
caused by Escherichia coli and other members of the Enterobacteriaceae
A protozoal parasite and a human pathogen
A flagellated protozoan parasite that colonizes and reproduces in the small intestine
Integration of the bacteriophage nucleic acid into the host bacterium’s genome
Process by which DNA is transferred from one bacterium to another by a virus
Exist within the bacterial cell by lysogeny, within a bacterial cell as a circular DNA in that it exists by replicating as if it were a plasmid and does not cause cell death, or can promote cell lysis during growth resulting in host cell death
Occurs when a cell bursts due to osmotic imbalance that has caused excess water to move into the cell
A test which uses antibiotic-impregnated disks to test whether particular bacteria are susceptible to specific antibiotics
Use dilution test
Stepwise dilution of a substance in a solution
A measure of the bactericidal activity of a chemical compound in relation to phenol
Infections that are a result of treatment in a hospital or a healthcare service unit
Adverse effects or complications caused by or resulting from medical treatment of advice
a reference to adjust the turbidity of bacterial suspensions so that the number of bacteria will be within a given range.
A substance that kills bacteria or slows their growth
A substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, or protozoans
Minimum Inhibitory Concentration- the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation
Disk diffusion test
Means of measuring the effect of an antimicrobial agent against bacterial grown in a culture
Minimum Bactericidal Concentration- the lowest concentration of antibiotic required to kill the germ
A class of medication used specifically for treating viral infections
Morbidity & Mortality Weekly Report.a weekly epidemiological digest for the United States published by the Centers for Disease Control and Prevention (CDC). It is the main vehicle for publishing public health information and recommendations that have been received by the CDC from state health departments.
a carrier, especially the animal (usually an arthropod) that transfers an infective agent from one host to another.
any inanimate object or substance capable of carrying infectious organisms, such as germs or parasites, and hence transferring them from one individual to another.
a protein produced by the body's immune system when it detects harmful substances, called antigens.
part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. The classical signs are pain, heat, redness, swelling, and loss of function.
the process of engulfing a solid particle
a poisonous substance produced within living cells or organisms