Microbiology Lab
Catalase negative
No bubbles =
Oxidase negative
None (Does not turn blue or have color change) =
False
Open toe shoe are acceptable lab footwear
A substance that tends to bring about oxidation by being reduced and gaining electrons.
Oxidizing agent
KOH Test
Purpose: Confirms Gram stain based on whether 3% KOH can lyse bacterial cell wall and release DNA.
Oxidase Test
Purpose: Differentiates microorganisms based on the presence of the enzyme cytochrome Coxidase in bacteria.
BHI with 6.5% NaCl
Purpose: Differentiates microorganisms based on their ability to grow in 6.5% NaCl. Interpretation: Positive: Growth (turbidity) Negative: No growth (clear)
Bile Esculin Hydrolysis
Purpose: Differentiates microorganisms based on their ability to hydrolyze esculin to esculetin and dextrose. The esculetin reacts with ferric citrate in the medium to form a dark brown-black complex. Interpretation: Positive: Blackening of the tube Negative: No color change or less than 1⁄2 of the slant is black
Catalase Test
Purpose: Differentiates microorganisms based on their ability to produce the catalase enzyme. Catalase breaks hydrogen peroxide down to water and oxygen. (Caution: Too much hydrogen peroxide will "water down" the reaction and give a false negative result.)
Nitrate Reduction
Purpose: Differentiates microorganisms based on their ability to reduce nitrate to nitrite or ammonia or nitrogenous gas. Interpretation: Positive: Red coloration after the addition Nitrate A and B reagents OR No color change after the addition of zinc Negative: Red coloration after the addition of zinc
Phenol Red Carbohydrate Tubes(Arabinose, Lactose, Trehalose, Mannitol, Sucrose, Xylose, etc.)
Purpose: Differentiates organisms based on carbohydrate fermentation. This media contains specific carbohydrates and the pH indicator phenol red. When an organism is able to ferment the carbohydrate present, an acid or an acid and gas are produced and the indicator changes from red to yellow. If the carbohydrate is not fermented, the color will remain red. Interpretation: Positive: Yellow color(fermentation) Negative: Red color
bacteria with a rod-shaped morphology.
Rods (bacilli)
ex. Bacillus
Rods may occur in chains...
ex. Pseudomonas aeruginosa
Rods may occur singly...
(counter stain, pink/red)
Safranin
Allow only certain microorganisms to grow and will inhibit the growth of other microorganisms.
Selective medium
Is another differential medium. Some species of Streptococcus and Enterococcus produce exotoxins (poisons) that completely lyse(destroy) red blood cells in blood agars.
Sheep blood agar (SBA)
brightfield microscopy
Simplest form of microscopy where light is either passed through or reflected off of a specimen?
corkscrew rods ex. Campylobacter
Spiral/Curved
simple stain
Staining with a single stain Allows microbiologists to visualize cell morphology, cell arrangement, and internal storage materials.
97% of the strains have proven to be coagulase positive
Staphylococcus aureus
Adding Gram's iodine to a starch agar plate. Iodine complexes with the starch macromolecule and causes the starch agar to turn black/brown. However, if the starch in the medium has been degraded by amylase produced by the bacteria, the starch agar adjacent to the bacterial growth will be clear after the addition of the iodine rather than black/brown.
Starch hydrolysis is detected by...
Catalase
Streptococcus and Enterococcus lack this enzyme but Staphylococcus includes this enzyme...
Bacteria that cannot grow in the presence of oxygen. Lack catalase.
Strict anaerobes
cleaning of a microscope
Take one sheet of lens paper (in your drawer) and wipe down Oculars Stage Objectives (10X, 40X, 100X)
aseptic technique
Taking precautions to prevent the contamination of others, yourself, your surroundings, and your lab supplies is known as...
a replacement for the Gram stain technique.
The KOH test IS NOT...
VERIFY the Gram stain technique.
The KOH test should only be used to...
The esculetin reacts with the ferric citrate to produce a dark brown or black phenolic iron complex turning the medium dark brown or black. However, the test is interpreted as a positive result only if more than half the medium is dark brown or black after incubation.
When an organism hydrolyzes the esculin to form esculetin and dextrose....(Bile esculin agar test)
sharps container
Where are slides, pipettes, and swabs are discarded?
Bile esculin agar
a selective and differential medium which is used to presumptively identify Enterococcus and group D Streptococcus based on the ability of an organism to hydrolyze esculin.
The coagulase test
excellent definitive test for confirming identification of Staphylococcus aureus It involves inoculating a small tube of blood plasma with a loopful of the organism and incubating it in at 37°C for 24 hours. If the plasma coagulates (clots), the organism produces coagulase and is considered coagulase positive. It should be emphasized that this test is valid only for Gram positive, staphylococcus-like bacteria because some Gram negative rods, such as Pseudomonas, can cause a false-positive reaction. However, the mechanism of clotting in such organisms is not due to coagulase.
microorganisms are everywhere
"Ubiquity of microorganisms" means...
Aerobic bacteria
(oxygen required for growth) grow by respiration, they use oxygen as a terminal electron acceptor, converting it to water. Produce Catalase.
Anaerobes that tolerate oxygen. Lack catalase.
Aerotolerant bacteria
Partially breakdown red blood cells producing a greenish discoloration around the colonies
Alpha hemolysis
a larger branched polymer of glucose.
Amylopectin
a straight chain polymer of 200 to 300 glucose molecules
Amylose
Into media to create selective conditions for growing specific microorganisms
Antibiotics, antifungals, dyes, and various inhibitory compounds are incorporated...
Microbes that live in extreme environments
Archaea
A set of specific practices and procedures performed under carefully controlled conditions with the goal of minimizing contamination by microbes.
Aseptic technique
Microorganisms not known to cause disease in healthy adults
BSL 1
Indigenous microorganisms that can lead to diseases of varying severity in healthy adults
BSL 2
Indigenous or exotic microorganisms that cause serious or potentially lethal disease through respiratory transmission
BSL 3
Microorganisms that are dangerous and exotic with high risk of aerosol transmitted infections. Rarely are there treatments or vaccines for these microorganisms, and the diseases they cause are frequently fatal
BSL 4
Produce amylases that degrade the starch molecule into molecules of maltose, glucose, and dextrins that can enter the bacterial cell.
Bacteria that hydrolyze starch...
18-24 hours old for best staining results.
Bacterial cultures to be Gram stained must be...
Too large to enter the bacterial cell, so some bacteria secrete exoenzymes to degrade starch into subunits that can then be utilized by the organism.
Because of their large size, starch molecules are...
Smear Preparation
Begins every staining procedure. Always come from a solid media, such as a plate or slant. Consist of a mixture of sterile water and bacteria on a slide. *Most common mistake is when placing to many bacteria on the slide.
Complete lysis of red blood cells around a colony and results in a clear zone surrounding the colonies.
Beta hemolysis
BSL 4
Biosafety level designation used to describe microorganisms that are dangerous and exotic with high risk of aerosol transmitted infections. Rarely are there treatments or vaccines for these microorganisms, and the diseases they cause are frequently fatal.
Oxidase positive
Blue =
Catalase positive
Bubbles =
Hydrogen peroxide (H2O2)
By product of respiration conducted by aerobic bacteria, and a highly reactive oxidizing agent that can damage enzymes, nucleic acids, and other essential molecules in the bacterial cell.
Enzyme which degrades hydrogen peroxide into harmless oxygen and water.
Catalase
Aerobic Staphylococcus from aerotolerant Streptococcus and Enterococcus, which lack this enzyme.
Catalase production can be used to differentiate...
Focus at 10X before moving to 40X
Coarse adjustment
bacteria with a spherical morphology
Cocci
ex. Streptococcus
Cocci may occur in chains...
ex. Staphylococcus
Cocci may occur in clusters...
ex. Micrococcus
Cocci may occur in tetrads (groups of four)...
ex. Neisseria
Cocci may occur singly, in pairs...
(primary stain, purple)
Crystal violet
the use of an artificial electron acceptor, N,N,N',N' -tetramethyl-p-phenylenediamine, which changes from yellow to blue when electrons are transferred from reduced cytochrome c to the artificial acceptor. The artificial acceptor is somewhat unstable and can oxidize if left exposed to air for prolonged periods of time. That being said, 30 seconds is the cut off time for the color change to occur and results to be interpreted. This is because oxygen in the environment can create a false positive result if results are read after this 30 second cut off.
Cytochrome oxidase is detected by...
Bacteria that carry out respiration where oxygen is the terminal electron acceptor; hence, the test differentiates between those bacteria that have cytochrome oxidase and use oxygen as a terminal electron acceptor from those that can use oxygen as a terminal electron acceptor but have other types of terminal oxidases.
Cytochrome oxidase occurs in...
Hans Christian Gram
Danish physician Attempted to perfect a staining technique that would differentiate bacteria from the eukaryotic nuclei of lung tissue, but in his research, he noticed that some bacteria retained the stain (crystal violet) appearing dark blue to purple while others did not.
Differential stain
Difference in stain uptake due to cell wall composition
Contains substances that cause some bacteria to take on an appearance that distinguishes them from other bacteria
Differential medium
(decolorizer)
Ethanol
Bacteria that can grow by respiration or fermentation-with or without oxygen. Produce Catalase.
Facultative anaerobes
Focus at 40X Add ONE drop of immersion oil to the slide before trying to view on 100X
Fine adjustment
Species of bacteria that do not exhibit any hemolysis of blood and have no effect on the red blood cells in a blood agar plate.
Gamma hemolysis
Have a thin layer of peptidoglycan (do not retain crystal violet)
Gram negative bacteria
will lyse in the KOH solution, releasing their DNA and causing the liquid to become very viscous. Often, "strings" of DNA can be seen adhering to the loop as it is raised from the slide.
Gram negative cells in a KOH test...
Have a thick layer of peptidoglycan (retain crystal violet)
Gram positive bacteria
Do not undergo lysis in the KOH solution and hence an increase in viscosity or DNA strings will not be seen in these cells.
Gram positive cells in a KOH test...
It is the first step used to identify bacteria.
Gram staining is a very important technique to learn because....
The mordant that "plugs" the pores in Gram positive cell walls, so the ethanol decolorizer, in step 6, will not wash it out.
Gram's Iodine
acid is usually produced
If an organism can breakdown carbohydrates by oxidation or fermentation...
Selects for the genus Staphylococcus (and other sodium tolerant organisms) but inhibits the growth of bacteria that cannot tolerate the salt concentration.
Incorporation of sodium chloride into mannitol salt agar (MSA)...
The deciding factor in whether a bacterium will retain crystal violet or not....
Is the amount of peptidoglycan present in the bacterium's cell wall.
KOH negative = Gram positive
KOH results (no strings present)
KOH positive = Gram negative
KOH results (strings present)
dark field microscope
Light Microscope Contrast is created by a bright specimen on a dark background. Reveals morphology and external structures. Stains are NOT required
phase-contrast microscope
Light Microscope Converts the differences in optical density (refractive index) of cells into different shades of brightness. Allows for visualization of morphology, external structures, and some internal structures. Stains are NOT required
bright field microscope
Light Microscope Simplest form of microscopy where light either passes through or reflects off a specimen. Requires the use of stains to visualize cells . Simple stain . Gram stain
fluorescent microscope
Light Microscope Uses high intensity illumination to excite fluorophores. Fluorescence (witnessed light)emanates from the sample (not illumination)
the KOH test requires a culture no older than 24 hours to be valid. Old Gram positive cultures will give a Gram negative result.
Like all other tests performed in the General Microbiology Lab...
flaming them using a Bunsen burner
Loops are sterilized by...
Biosafety levels 1 and 2
The organisms used in the General Microbiology lab fall under which biosafety levels?
phenol red, from red at alkaline pH values to yellow at acidic pH values.
The presence of acid is indicated by a color change in the pH indicator...
amylose and amylopectin
The starch macromolecule consists of two constituents:
cytochrome oxidase
This enzyme catalyzes the transfer of electrons from reduced cytochrome C to molecular oxygen, producing oxidized cytochrome C and water.
Counter stain (safranin) is added to give them a pink to red appearance
To view the Gram negative bacteria...
Inhibit the growth of Gram positive organisms other than Enterococcus and group D Streptococcus
What does oxgall (bile salts), do in Bile esculin agar?
objectives
What is the name of the microscope lens that have the magnifying powers of 10X, 40X, and 100X?
lens paper
What type of paper is used to clean your microscope?
It ferments mannitol producing acid and changing a pH indicator in the media from red to yellow around the colonies. Some other species of Staphylococcus and staphylococci (organisms similar to Staphylococcus) cannot ferment mannitol, and their growth on MSA results in the agar remaining red around the colonies.
When Staphylococcusaureus grows on mannitol salt agar (MSA)...
