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Microbiology Lab Practical I


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This is the light source for the microscope. It is found in the base of the microscope.
This is a two lens system that collects and concentrates light from the illuminator, then directs the light into the Iris diaphragm.
Abbe condenser
This small lever found on the condenser regulates the amount of light that enters into the lens system.
Iris diaphragm
A platform used to place the slide on. It has a hole in the center that allows light from the illuminator to pass through, thus lighting the specimen on the slide.
Mechanical stage
The revolving part that contains the objectives
What you look through
Oculars or eyepieces
Enlargement of specimen
The ability of the lens to show two adjacent objects as distinct entities
Resolving power
The bending power of light
Refractive Index
This allows the image to remain largely in focus, with only minor adjustment needed when switching from one objective lens to another.
Par focal
Always multiply the objective lens (4X, 10X, 40X or 100X) value by the ocular lens value (10X) to give you the total magnification by which you are viewing the specimen
Total magnification
A more positively charged colored dye will have an affinity for the negatively charged components of the cell it will stain. Dyes are cationic.
Examples: Methylene blue, crystal violet, carbol fuschin
Basic Stains
A more negatively charged colored dye will have an affinity for positive components of the cell it will stain. Dyes are anionic.
Examples: Picric acid
Acidic Stains
What is the purpose of heat-fixing specimens?
The purpose of heat-fixing specimens is to allow the proteins within the cell wall to coagulate so the organism will stick to the glass slide.
A method of staining that differentiates between Gram (-) and Gram (+) bacteria. The results of this stain depend upon the cell wall of the bacterium.
Gram Stain
Name Gram Stain's 4 reagents in the following order:
Primary Stain
Decolorizing Agent
Primary Stain - Crystal Violet
Mordant - Gram's Iodine
Decolorizing Agent - 95% Ethanol
Counterstain - Safranin
In Gram Staining, what is the purpose for the primary stain, Crystal Violet?
All cells will stain purple/violet in color
What is the purpose for the mordant, Gram's Iodine?
The iodine will bind with the Crystal Violet to form a CV-I complex that locks within the peptidoglycan of the bacterium (cell wall).
When Gram Staining, what is the purpose for the decolorizing agent, 95% Ethanol?
The ethanol will perform 2 actions:

i. It is a lipid solvent, so the outer membrane of the Gram negative bacteria will melt away and washes out the CV-I complex, leaving the Gram negative bacteria colorless.

ii. It will dehydrate proteins within the Gram positive cells which close the pores of the cell wall, locking in the CV-I complex.

Decolorizing is the most critical step of Gram staining.
When Gram Staining what is the purpose for the counterstain, Safranin?
Because the outer membrane of the Gram negative bacteria has been melted away and the CV-I has been washed off, safranin can stain these cells. These will stain pink.

Gram positive cell pores have been sealed shut, so safranin cannot enter into these cells. These will remain purple/violet.
Cultures that are old (more than 48 hours) will lose their ability to maintain the crystal violet stain and will decolorize very easily - what is the name given to these cultures?
Gram variable - These cultures are distinguishable because the morphology is consistent throughout the microscopic field of view, but the Gram result is not. You would see some that are Gram negative and some that are Gram positive.
What is a substance that is produced within the cytoplasm of the bacterium and secreted to the outside of the cell wall where it encapsulates the bacterium. It is generally composed of mucoid polysaccharides, polypeptides, or glycoproteins.
What is the purpose of the Capsule?
Enhance virulence
Capsule stains must NOT be heat-fixed or washed with water.
Capsules are difficult to stain, so the background will be stained. This is called?
Negative Stain
What are the two reagents used in Negative Staining?
Crystal Violet and 20% Copper Sulfate
What is the primary stain in Negative Staining?
Cystal Violet
What is the decolorizing agent in Negative Staining?
20% Copper Sulfate
What is the counterstain in Negative staining?
20% Copper Sulfate
What is the purpose of the Crystal Violet in Negative Staining?
Everything on the slide will be stained purple.
What are the purposes for the 20% Copper Sulfate in Negative Staining?
a. 20% CuSO4 decolorizes by removing the crystal violet

b. Cells will take up the copper sulfate, while the background will remain violet/purple.
Name a differential stain used to detect cells capable of retaining a primary stain when treated with acid alcohol. The acid-alcohol cannot decolorize the cells.
Acid-fast stain
Acid-Fast Stain is important for the identification of which bacteria?
Mycobacterium synthesize an outer molecule composed of a thick waxy (lipid) substance called?
Mycolic acid

Mycolic acid helps protect against the immune system, but also repel stains.
Name two members of the genus Mycobacterium?
Mycobacterium tuberculosis and Mycobacterium leprae
What is required to remove the thick waxy mycolic acid?
What is the primary stain used in Acid-Fast Stains, and what is its purpose?
Carbol Fuschin

This hot pink stain is lipid soluble and the heat will help it penetrate through the mycolic acid
What is the decolorizing agent used in the Acid-Fast Stains, and what is its purpose?
Acid Alcohol

This will decolorize everything on the slide except acid-fast bacterium
What is the counterstain used in the Acid-Fast Stain, and what is its purpose?
Methylene Blue

Only non-acid-fast organisms present will stain blue
A differential stain used to detect the presence and location of spores.
Endospore stain
Name two generra that produce spores?
Clostridium and Bacillus (Gram + rods).
Name the 3 reagents used in endospore staining.
Malachite Green
Tap Water
Name the primary stain used in endospore staining, and its purpose.
Malachite green

Malachite green must be applied with heat to penetrate the multiple protein coats surrounding the spores

The endospore and vegetative part of cell will accept the primary stain
Name the decolorizing agent used in endospore staining, and its purpose.
Tap water

Because malachite green is water soluble, any excess malachite green will be washed away, but stain within the spore will not be removed

Stain within the vegetative cells will be washed away, leaving them colorless
Name the counterstain used in endospore staining, and its purpose.

Vegetative cells will now accept the safranin and be stained red/pink

Endospores will remain green or clear
What is the production or formation of spores called?
As favorable conditions return, the free spore will germinate back to what kind of a cell?
Vegetative Cell
Microbiologists use the term _______ to refer to an increase in the population of microbes, rather than size.
The result of the increase in population is a ________ an aggregation of cells arising from a single parent cell.
Name the organisms that utilize an inorganic carbon source (i.e. CO2) as their sole carbon source. They make organic compounds from CO2 and don’t need to feed on organic compounds from other organisms to acquire C.
Organisms that catabolize reduced organic molecules (i.e. proteins, carbs, amino acids, fatty acids) that are acquired from other organisms
Organism that acquire energy from redox reactions involving inorganic and organic chemicals
Organisms that use light as their energy source
Organisms that acquire electrons or H atoms from inorganic sources (i.e. H2, NO2-, H2S, Fe2+) are called ________?
Organisms that acquire electrons (as part of hydrogen atoms) from the same organic molecules that provide them Carbon and energy are also called ________?
Oxygen is necessary for this organism because it serves as the final electron acceptor of the ETC
Obligate aerobes
Oxygen is deadly for this group of organisms because it is an oxidizer and will steal electrons away from the ETC.
Obligate anaerobes
Aerobic organisms that can maintain life via fermentation or anaerobic respiration, though their metabolic efficiency is often reduced in the absence of oxygen. Ex. Escherichia coli
Facultative aerobes
These organisms do not use aerobic metabolism, but tolerate oxygen by having some enzymes that detoxify oxygen’s poisonous forms. Ex. Lactobacillus
Aerotolerant aerobes
Aerobes that require oxygen levels of 2-10%. These types of bacteria have limited ability to detoxify H2O2 and superoxide radicals. Ex. Helicobacter pylori
Molecular oxygen with electrons to boost it to a higher energy state. It is highly reactive (oxidizing) and is used by certain WBCs to kill pathogens.
Singlet oxygen
Reactive and toxic, some aerobic organisms will produce an enzyme called superoxide dismutase to detoxify it. Anaerobes are susceptible to oxygen because they do not produce a superoxide dismutase enzyme.
Superoxide radical
An enzyme that catalyzes the decomposition of a superoxide into hydrogen peroxide and oxygen.
Superoxide dismutase
If an organism lacks the catalase, peroxidase and superoxide dismutase enzymes, it will be susceptible to ________?
Peroxide anion
Enzyme breaks down H2O2 without forming O2, using a reducing agent such as the coenzyme NADH:
Name this radical that results from the incomplete reduction of H2O2. This is the most reactive of the 4 forms.
Hydroxyl radical
The conversion of atmospheric nitrogen into compounds, such as ammonia.
Nitrogen fixation
___________ is often the growth-limiting nutrient.
________ is acquired from organic and inorganic nutrients. For example, all cells recycle _________ from their amino acids and nucleotides.
95% of the dry weight of cells is made up of what 4 chemicals?
Carbon, Nitrogen, Hydrogen, and Oxygen

(The rest is made up of P, S, Mn, Mg, Cu, Fe and a few others called trace elements)
____________ such as vitamins, amino acids, purines, pyrimidines, cholesterol, NADH, and heme are elements that some organisms cannot manufacture themselves. These growth factors must be acquired from another source.
Growth factors
Name three physical requirements of microorganisms.
Physical Effects of Water
Proteins ________ when they lose their three-dimensional structure or "conformation") and thus their characteristic folded structure.

Temperature – affects the three-dimensional configuration of biological molecules. Specifically, proteins require proper folding into a three-dimensional shape to function properly. This folding is determined in part to temperature-sensitive H-bonding which form at lower temps, and break at high temps. When the bonds break, the proteins unfold (denature), and functionality is lost. Therefore, temp regulation is important in microbial growth.
What is the lowest temperature at which an organism is able to conduct metabolism. Bacteria can survive, but the metabolic activity is affected in a negative way.
Minimum growth temperature
What is the highest temperature at which an organism continues to metabolize. If the temperature exceeds this value, the proteins are permanently denatured. When the proteins are denatured, functionality is lost and the organism will die.
Maximum growth temperature
Temperature at which an organism’s metabolic activities produce the highest growth rate.
Optimum growth temperature
An organism survives over a range of temperatures, which spans from the minimum to maximum growth temps.
Temperature range
Organisms that grow best at temps below 15 degrees C. These organisms grow in ice, snow, etc.
Organisms that grow best at temps ranging from 20 degrees C to 40 degrees C though they can survive at higher and lower temps.
Organisms are mesophilic organisms that can survive brief periods of high temps. Examples would be organisms that cause spoilage in pasteurized and canned food.
Organisms that grow at temps above 45 degrees C in environments such as compost piles and hot springs.
Some members of the Archaea grow at temps above 80 degrees C or even above 100 degrees C. These types of bacteria generally do not cause human disease.
Give the expression pH.
What is meant by pH?
Range of numbers expressing the relative acidity or alkalinity of a solution. In general, pH values range from 0 to 14. The pH of a neutral solution, i.e., one which is neither acidic nor alkaline, is 7. Acidic solutions have pH values below 7; alkaline, or basic, solutions have pH values above 7.
What bacteria prefer a neutral pH (around 6.5-7.5)?
What bacteria prefer an acidic environment (less than 6.5)?

Helicobacter pylori
________ conditions can inhibit growth of microbes, but there are microbes that prefer these conditions
Bacteria that prefer alkaline conditions?

Ex. Vibrio cholerae
Microbes that are adapted to growth under high osmotic pressure.
Obligate halophiles

Ex. Organisms that live in Great Salt Lake
Microbes who do not require high salt concentrations, but can tolerate them.
Facultative halophiles

Ex. Staphylococcus
Organisms that live under extreme pressure.

Hydrostatic Pressure

Ex. Organisms that live in ocean basins and trenches
Organisms don’t live independently, they live in association with each other, whether they are the same or different species. The name given to such associations is?
Ecological Associations
Relationships in which one organism harms or even kills another organism.
Antagonistic relationships

Ex. viruses
Individual members of an association cooperate so that each receives benefits that exceed those that would result if each lived by itself. This is called?
Synergistic relationships
Organisms live in such close nutritional or physical contact that they become interdependent. This is called?
Symbiotic relationship
Complex relationships among numerous individuals, often of different species. These individuals attach as groups to surfaces and then display metabolic and structural traits different than from those expressed by any microbe alone. These are referred to

It is possible that up to 65% of bacterial diseases are caused by biofilms.

Ex. Dental caries
This process is a response of bacteria in which they “sense” how many other bacteria are in the environment. The bacteria will secrete molecules into the environment that act as signals.
Quorum sensing
A sample of a microorganism
Collection of nutrients specifically designed to grow bacteria.
Microorganisms that grow from an inoculum OR the act of cultivating microorganisms
Cultures composed of cells arising from a single progenitor, colony-forming units (CFUs)
Pure culture
Free of microbial contaminants
Liquid culture media
Semi-solid media that contains a complex polysaccharide.

Agar is indigestible by microbes, will dissolve into water at 100 degrees C and solidify below 40 degrees C. Agars can be poured into petri plates, placed into tubes that are cooled on slants or kept vertical to produce deeps
Medium in which the exact chemical composition is known.
Defined Media (synthetic media)

Often used to grow fastidious organisms.
Medium that contains nutrients that released by partial digestion of yeast, beef, soy or proteins.
Complex media
Name advantages of Complex media over defined media.
Supports a wider variety of different microorganisms

Used to culture organisms whose exact nutritional needs are unknown

Ex. Nutrient broth, TSA, MacConkey agar, blood agar
This type of media typically contains substances that either favor the growth of particular microorganisms or inhibit the growth of unwanted ones.
Selective Media
Name an example of selective media.
1. Eosin, methylene blue, crystal violet dyes, bile salts are included to inhibit growth of Gram-positive bacteria without affecting Gram-negatives.
This media is formulated so that either the presence of visible changes in the medium or differences in the appearances of colonies helps differentiate among different kinds of bacteria.
Differential Media
This media contain compounds that chemically combine with free oxygen and remove it from the medium.
Reducing media

Ex. Sodium thioglycollate broth (Anaerobic Media)
Special media used to handle clinical specimens when transporting urine, feces, saliva, sputum, blood, or other fluids to ensure that contamination is limited and infection is inhibited.
Transport Media
Bacteria reproduce by _________?
Binary fission
Binary fission produces _________ growth.
The time required for a bacterial cell to grow and divide.
Generation time
The _____________is a graph that plots the numbers of organisms in a growing population.
Growth curve
This phase is when the cells are adjusting to their new environment. The cells will not automatically reproduce, but will synthesize enzymes to utilize the nutrients.
Lag Phase

This phase can last for an hour or for days.
During this phase the bacteria will start synthesizing the chemicals to conduct metabolism in their environment. Then they enter a phase of rapid chromosome replication, growth, and reproduction. The bacterial population increases logarithmically.
Log Phase

At this stage, the bacteria are more susceptible to antimicrobial drugs and these bacteria are better for Gram staining.
Because the nutrients are becoming depleted and wastes accumulate, and reproduction decreases, cells begin to die at this stage and the number of dying cells equals the number of cells being produced.
Stationary Phase
Because nutrients are not being added and wastes are not being removed, the population will die at a rate faster than they are produced. This is the ________ phase?
The clinical term for dental cavities is?
Dental caries
In a person’s oral cavity, there are many bacteria; however, there are 3 that have a tendency to cause dental cavities. Name these three?
Lactobacillus acidophilus
Strepotococcus mutans
Actinomyces odontolyticus
Lactobacillus acidophilus,
Strepotococcus mutans,
Actinomyces odontolyticus all will ferment carbohydrates that you ingest. When fermented, an end product of Lactic acid is produced, as well as a sticky molecule called _________?
Dextran molecule holds the lactic acid directly against the teeth in a biofilm layer, allowing the acid to break down the calcium and enamel on your teeth⬦a process called?
Enamel decalcification begins with a pH of _____ and accelerates when the pH lowers to towards _____.
5.5, 4.4

If these molecules are not removed by brushing, you will develop caries.
What test measures the amount of acid produced by the action of lactobacilli on glucose.
Snyder Test
To test for the presence of lactobacilli, we will use a differential medium called
Snyder Agar
What is the substrate for the Snyder Agar?
What is the pH indicator for the Snyder Agar?
Bromcresol green
What occurs to the media to suggest susceptibility to dental caries?
Yellow color change within 2-48 hrs

Cultures containing lactobacilli from saliva will show glucose fermentation with the production of acid. This will lower the pH level within the agar, thus reacting with the pH indicator. This will cause a color change to yellow.

No color change is indicative of lower susceptibility.
What are the micro-organisms which ordinarily grow on the various surfaces animal?
Normal flora
Name 5 microorganisms that make up the normal flora of the skin.
Name 4 microorganisms that make up the normal flora of the eye (conjunctiva).
Name some microorganisms that make up the normal flora of the upper respiratory tract.
Name 3 microorganisms that make up the normal flora of the mouth and teeth.
Anaerobic spirochetes
fusiform bacteria
Name some of the microorganisms that make up the normal flora of the intestinal tract - start with upper intestine - then lower intestine.
Upper intestine – lactobacilli and enterococci.

Lower intestine and colon - 96-99% composed of anaerobes such as members of the genera Bacteroides, Lactobacillus, Clostridium and Streptococcus, and 1-4% composed of aerobes, such as coliforms, enterococci, and members of genera Proteus, Pseudomonas, and Candida
Name some microorganisms that make up the normal flora of the genitourinary tract.
gram negative enteric bacilli
protozoa such as Trichomonas
This type of media is used to cultivate Streptococcus species. This organism will demonstrate different types of Hemolytic patterns
Blood agar
This type of media is used to cultivate Neisseria species.
Chocolate agar
Oxidase Reagent is also called ________?
What occurs when an Oxidase Reagent is dropped onto a bit of growth on the Chocolate agar plate?
There should be an area that turns purple-black, this is a positive result, indicating only that a species of Neisseria is present.
This type of media is used to demonstrate the presence of the genus Diphtheroids.
Mueller-Hinton tellurite
The black coloration when testing for Diphtheroids using Mueller-Hinton tellurite is due to the diffusion of the _______________into the bacteria, where it is reduced into tellurium metal and then precipitates inside the cells.
Tellurite ions
What type of media is used to cultivate molds and yeasts?
Sabouraud Agar

Yeasts will be mucoid on the surface of this agar while molds will be fuzzy with some coloration.
This infection occurs when bacteria move up the ureters from the bladder to the kidneys.
This infection limited to the ureters and kidneys.
This is inflammation that results in renal corpuscle damage.
This is inflammation of vagina, can be caused by a protozoan, Trichomonas vaginalis.
This is inflammation of bladder, and can be caused by a pathogenic fluke, Schistosoma haematobium.
Bladder infections
This is bacteria in the urine.

Pathological when the colony count is >100,000 bacteria/mL of urine.
Name some bacteria that can cause an urinary tract infection.
Staphylococcus aureus
Enterococcus faecalis
Proteus mirabilis
Proteus vulgaris
Pseudomonas aeruginosa
Name a protozoa that can cause an urinary tract infection.
Entamoeba histolytica
Name a virus that can cause an urinary tract infection.
Herpes Simplex Type II
Name a fungi that can cause an urinary tract infection.
Coccidioides immitis and Candida albicans
Name a helminth that can cause an urinary tract infection.
Wuchereria bancrofti
Give the equation to do a UTI colony count using a calibrated loop.
(number of colonies)*(factor that converts loop calibration to 1ml) = Organisms per ml

Loops come in 3 sizes: 0.1 ml, 0.01 ml, 0.001ml.

Incubate for 24 hrs at 37ËšC
Name 3 biochemical tests that can be used to identify Staphylococcus species.
Mannitol Fermentation
DNase production
Novobiocin Sensitivity.
Name the substrate for Mannitol Salt Agar.
Name the pH indicator for Mannitol Salt Agar.
Phenol red
Name the three ingredients to Mannitol Salt Agar.
7.5% NaCl
Mannitol (substrate)
Phenol red (pH indicator).
What species will grow on Mannitol Salt Agar?
All Staphylococcus species.

Enterococcus faecalis (member of Streptococcus family) will also grow and ferment mannitol.

The high salt concentration inhibits most bacteria from growing, however
How do you determine a positive fermentation reaction using Mannitol Salt Agar?
A positive fermentation reaction turns the media surrounding the growth yellow.
What Staphylococcus species will ferment Mannitol?
Staphylococcus aureus
Name the two ingredients in DNase Agar.
Polymerized DNA (substrate)
methyl green (color indicator).
Name the substrate in DNase Agar.
Polymerized DNA
Name the color indicator in DNase Agar.
Methyl green
_________, produced by some bacteria, is a hydrolytic enzyme that will degrade polymerized DNA.
What three bacteria will test positive for DNase Production?
S. aureus
S. intermedius
S. xylosus
When performing the DNase Production test, Methyl green will combine with ________?
Polymerized DNA
What media is used for Novobiocin Sensitivity testing?
Mueller-Hinton Agar
What property does Mueller-Hinton Agar have that allows for the diffusion of the antibiotic Novobiocin through the media in a constant manner.
Mueller-Hinton Agar is very porous
How do we determine if an organism is sensitive to Novobiocin when performing the Novobiocin Sensitivity test?
We look for a Zone of Inhibition.
What particular species is confirmed with the Coagulase Test?
Staphylococcus aureus
__________ DNA cannot bind the methyl green and the green color of the media will fade.

This is a positive test result.
Methyl green will combine with ___________ DNA.
_____________ species can be identified by reaction on the Blood agar plates due to their hemolytic patterns
Partial hemolysis of RBCs

This gives a greenish tint to the media surrounding the growth.
Complete hemolysis of RBCs

The will allow the media to have a clear zone around the growth
No hemolysis of RBCs

No change in media
Causes the destruction of red blood cells
Act on phagocytes
Can cause a Scarlet Fever Rash
Erythrogenic toxins
Hydrolyzes tissue cement
A fibrinolysin
Destroys viscous tissue debris and genomic material
Name five biochemical tests that are used to identify Streptococcal Species
Bacitracin & SXT Susceptibility Test
H2S production
MSA growth
C.A.M.P. Test
What media is Bacitracin & SXT Susceptibility Test performed on?
Blood Agar

Look for Zones of Inhibition
otherwise the organism is resistant
What media is used for the H2S production test?
Bile Esculin slant
Organisms positive for H2S production will hydrolyze the ___________ and the end product will react with the iron salts of the media.
Glycoside esculin
Mannitol Salt Agar is used to identify which Streptococcal species?
Enterococcus faecalis

Growth is the only reaction needed - Fermentation will also occur
What is observed on the Blood agar plates?
Name 4 Streptococcal species that will have Beta hemolysis.
Streptococcus agalactiae
S. equi
S. pyogenes
S. zooepidermicus
Name 3 Streptococcal species that will have Alpha hemolysis.
Streptococcus bovis
S. oralis
Enterococcus faecalis
What media is used when performing a C.A.M.P. Test?
Blood agar
What two organisms was the C.A.M.P. Test originally developed to distinguish between?
S. pyogenes and S. agalactiae
What organism is used to enhance the effects of the C.A.M.P. Test?
Staphylococcus aureus
Where the hemolysins from S. aureus and S. agalactiae come together, there will be a blending of hemolysins to produce an _______________ – shaped clear zone.
“Arrowhead effect”

C.A.M.P. Test
Streptococcus agalactiae is CAMP positive or negative?
CAMP positive
Streptococcus pyogenes is CAMP positive or negative?
CAMP negative
This type of media will “select for” specific bacterial groups.
Selective Media
This type of media will distinguish among morphologically and biochemically related bacterial groups, such as gram-negative rods.
Differential Media
What media contains certain chemicals that permit certain bacteria to grow, while inhibiting others.
Selective Media
The bacteria that grow on this type of media will incorporate ___________ from the media into their appearance and then produce a characteristic trait that will make them look different from other types of bacteria that grow on the same media.

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