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The streak plate technique uses spatial dilution (Increases the space b/t organisms) to create
______ means a specimen in focus under the low power objectives should be nearly in focus under high power objectives as well
Explain the difference in smear preparation when bacteria are taken from cultures in broth or solid agar media
A smear can be made directly from a broth culture since the bacteria are suspended in a liquid. In the case of solid agar growth, a loopful of water must first be placed on the slide. The inoculum of bacteria is mixed into this drop of water and the suspension formed is spread on the slide to form a bacterial smear.
Name two results of heat fixation
Two results of heat fixation are killing the bacterial cells and adhering them to the surface of the slide.
Explain the chemistry of staining a bacterial cell
Dyes are salts. They have a chromophore group which is either positively or negatively charged. The chromophore group combines with the oppositely charged groups in bacteria to stain the cells.
Describe the color and shape of the stained cells. Draw representative cells.
Crystal violet stains cells violet, while safranin stains cells pink. Bacillus mycoides is a medium size bacillus that appears in long chains. Staphylococcus aureus is a coccus that appears as single cells, diplococci, and irregular clusters of cocci. Escherichia coli is a short bac illus that appears as single cells or as diplobacilli.
A simple stain shows
cell morphology (shape) and arrangement and does not distinguish between different types of bacteria (like G+ and G-)
Resolving power =
the ability of a microscope to distinguish two closely spaced objects as being distinct from each other.
Indicate the relationship b/t numerical aperture and resolving power in the oil immersion lens
The numerical aperture is highest with the oil immersion lens. As the numerical aperture of the lens increases, the resolving power increases.
Working distance =
The working distance is the distance between the slide and the objective lens. The greater the magnification of a lens, the shorter is the working distance.
Indicate the effect a reduction of illumination has on resolving power
A reduction in illumination reduces the resolving power.
Bacillus anthracis is _____ shaped while Streptococcus pyogenes is _____ shaped
rod-like, circular clustered
Factors that affect microscope function include
resolving power, numerical aperture, magnification and working distance
Alpha vs. Beta hemolysis
Beta-hemolysis produces a clear halo around the colony on blood agar, while alpha-hemolysis produces a green halo around the colony.
What is the purpose of flaming during any transfer?
The purpose of flaming is to eliminate any source of contamination so that a pure culture can be transferred.
2 methods of inoculation completed with an inoculating loop
An inoculating loop can be used to streak the surface of a solid agar medium in a slant or petri dish. It can also be used to transfer a loopful of broth culture to another broth medium.
Autoclave or steam sterilization
Uses steam under pressure and is sterilization of culture media, reagents, and laboratory tools
Uses an oven to generate hot dry air
Sterilization of glassware and moisture sensitive preparations
Uses an open flame to incinerate microorganisms
Sterilization of tools like inoculating loops/needles
Aseptic technique is used to:
establish and maintain pure cultures
prevent contamination of established cultures and the environment
Key step in loop dilution procedure that favors isolation of pure cultures
Transferring a loopful of the culture from the broth culture to molten agar tube 1 to tube 2 to tube 3 serves to dramatically reduce the numbers of cells in the second and third tubes so that colonies of pure cultures can grow on the agar after pour plates are made.
Why does MacConkey agar favor growth of gram neg. bacteria?
MacConkey agar contains bile salts which is strongly inhibitory to gram-positive cells. Only gram-negative fermenters or nonfermenters of lactose grow on this medium. Lactose fermenters produce red colonies, while lactose nonfermenters produce colorless or white colonies.
Advantage of specialized media
Specialized media can be used to favor the growth of distinct microorganisms to the exclusion of all others.
Why does E. coli appear red on MacConkey agar?
Escherichia coli ferments the lactose in this medium to acids and the neutral red indicator turns red in the presence of acid. The colonies turn red.
PEAB test is
Used to select for the growth of gram-positive cocci such as streptococci and staphylococci
Generally gram negative bacteria do not grow on it
Bile salts inhibit G+ bacteria and allow G- bacteria to grow
G- bacteria that ferment lactose to acids cause the indicator to change color to red, so that red or pink colonies appear.
Bacteria unable to ferment lactose (e.g., Salmonella) appear white or colorless
Mannitol salt agar
MSA is highly selective, and used for the isolation of staphylococci from mixed cultures
Staphylococcus aureus is osmotolerant
as members of mixed populations in and on the body and in inanimate environments
Selective and differential media and pure culture techniques like the streak plate and loop dilution can be used to
isolate specific kinds of microorganisms from both animate and inanimate environments
is a gelatinous covering around the outside of a cell that adheres tightly to the cell surface.
Function of capsule
interfere with phagocytosis. They promote attachment of encapsulated cells to membranes. Capsules also are more resistant to the lethal action of complement.
Why is the capsule procedure considered a neg. stain?
The capsule is not stained in this procedure. The background and the bacterial cell are stained.
Explain how the capsule of Klebsiella pneumonia may promote the development of severe necrotizing pneumonia
Klebsiella pneumoniae is gram-negative and is encapsulated. This organism is resistant to phagocytosis by macrophages and neutrophils in the lung and is also resistant to the lethal action of complement. This organism is able to spread in the lung and produce severe lesions.
How would a lab tech use capsules to identify different encapsulated bacteria?
The Quellung test results in swelling of the capsule in the presence of specific antibody and is a useful test for the identification of different encapsulated bacteria.
Capsules enhance virulence (the ability to cause disease) in a variety of ways:
Interfere with phagocytosis by macrophages
Promote infection by adhering to host cells
Gram negative encapsulated bacteria are more resistant to the lethal action of complement (causes lysis of cells) and other serum factors
Enhance resistance to lethal actions of heavy metals, dehydrating, and attack of bacterial viruses
Capsular swelling in the presence of specific antibody
Valuable laboratory test for the sero-identification of specific encapsulated bacteria
formation is not a method of cell division, are metabolically inactive, only one forms in a cell, heat is necessary to force the primary dye into an endospore
In a negative stain, the purpose of India ink is
to create a color contrast b/t the capsule and background light
A mordant is a chemical that forms a complex with the primary dye and the cell wall of the cell. The mordant binds the primary dye more tightly to the bacterial cell.
The decolorizing agent removes the primary dye from a cell so that the cell is colorless
The counterstain stains the decolorized cells with a different color than the primary dye so that one may distinguish between cells having Gram-positive or Gram-negative staining reactions by color alone.
Gram + vs. Gram -
The alcohol shrinks the pores of Gram-positive cells so the primary dye is retained in the cell. Gram-negative cells contain lipid in the cell wall. The decolorizing agent removes the lipid and the crystal violet-iodine complex from the cell wall so that the cell is unstained. The counterstain stains the Gram-negative cell.
Most important step in Gram stain
The most important step in the Gram stain procedure is the decolorizing stage. This step decolorizes Gram-negative cells so that they can be stained by the counterstain. Gram-positive cells retain the primary dye after d ecolorization with 95% alcohol.
Which organisms show endospores?
Gram-positive bacilli in the genus Bacillus or Clostridium show endospores.
What color would be seen if alcohol were left out of the Gram stain procedure?
All bacteria would stain Gram-positive and would be violet since no decolorizing agent was applied. The primary dye would bind to all the available receptors in the cells so that the counterstain could not bind to the cells.
The Gram Stain
is a useful diagnostic tool that divides bacteria into two groups (Gram-positive and Gram-negative) based on differences in their cell walls
It is inexpensive, rapidly performed, and offers evidence for the presence of specific organisms causing medical problems
The Gram reaction of bacteria is a fixed characteristic
Why is heat used in the acid fast stain?
Heat softens the waxy lipid layer and forces the first dye into the walls of the cell
Why are acid fast cells not decolorized despite the treatment with acid alcohol?
The primary dye is tightly bound to the waxy lipids in the cell wall of acid-fast cells. These waxy lipids prevent entry of the acid alcohol decolorizing agent into the cell.
An endospore is an oval, metabolically inactive structure that is formed within the bacterial vegetative cell.
The sporangium is the remnant of the bacterial cell that is present after endospore formation.
Germination vs. sporogenesis
Germination is the process concerned with the breakdown of the spore wall and the development of a metabolically active vegetative cell. Sporogenesis is the process by which an endospore is formed within the original vegetative.
Two genera of bacteria that produce endospores
Bacillus (Gram-positive, aerobic rods)
Clostridium (Gram-positive, anaerobic rods)
Location of endospores
The location of the spore in the vegetative cell does not change for a given bacterial cell
Clostridium tetani show spores located at the end of the cell (terminal)
Only one spore forms in a bacterial cell
2 functions of flagella
Flagella rotate and move cells towards more favorable environments and away from more harmful environments. The motion of flagella also helps pathogenic bacteria spread in the body.
3 techniques to demostrate motility and explain the results seen for each.
Three techniques to demonstrate the presence of flagella are the use of a prepared slide of flagella, inoculation of semi-soft motility agar and the hanging drop preparation. The flagella slide shows stained flagella extending from the cell surface. Motile organisms produce cloudiness in the motility agar on either side of the stab line. Hanging drop preparations show the random jiggling motion typical of brownian motion and the directed motion typical of true motility.
Observes differences in results for motile and nonmotile bacteria in motility agar medium
Motile bacteria use their flagella to swarm through the soft agar and produce cloudiness away from the original streak line. Non-motile bacteria lack flagella and only show growth along the stab inoculation.
Explain the influence of 02 on the observed results in motility agar medium and indicate which organisms is most influenced by O2
Motile bacteria such as Pseudomonas aeruginosa which are aerobes tend to swarm at the agar surface where oxygen is plentiful. They show less cloudiness inside the agar where the oxygen content is lower. Facultative bacteria such as Proteus vulgaris show swarming at the surface and swarming within the agar. Non-motile bacteria only show growth along the stab line at the surface and within the agar.
True motility vs. Brownian
True motility requires energy expenditure by the cell, Brownian motion results from kinetic energy of molecules around the cell
Motile bacteria can move toward favorable environments or away from unfavorable environments
When should an acid fast stain be completed?
An acid-fast stain should be completed where the cells in the Gram procedure are weakly stained or stain in a Gram-variable fashion.
Advantages to cells with capsules
Cells with capsules are more resistant to phagocytosis than non encapsulated cells. Cells with capsules can more readily attach to mucous membranes than cells lacking capsules.
Bacterial unknowns are identified by
type of growth on selective and differential media, differential staining procedures, and by other characteristics like motility, pigment formation, oxygen requirements, and capsule formation
Mesophiles grow at 20 C to 45 C. Escherichia coli and Staphylococcus aureus are mesophiles
Psychrophile vs. psychrotroph
Psychrotrophs are a special category of mesophile which can also grow at 0 C. Psychrophiles only grow at low temperatures.
Why are pathogenic bacteria commonly mesophiles?
Pathogenic bacteria grow at 37 C which is a mesophilic temperature.
Facultative vs. obligate thermophiles
Facultative thermophiles grow at 37 C, but prefer to grow at elevated temperatures. Obligate thermophiles only grow at the elevated temperatures.
At the max temp for growth, reproduction is reduced due to
protein denaturation, loss of enzyme function, and cell death
Explain why the solute proline is valuable to staphlyococci
A solute such as proline can be accumulated internally to raise the osmotic pressure. This compound causes the uptake of water by the cell and prevents plasmolysis found when the cells are immersed in hypertonic solutions.
Account for the growth of obligate halophiles on Halobacterium salt agar
Halobacterium salinarium is an extreme halophile. The high salt concentration stabilizes the enzymes in the cell and the cell wall. This organism accumulates potassium ions internally to prevent plasmolysis.
An environment external to the cell with higher solute concentration and lower water concentration
Plasmolysis results when the cell membrane shrinks from the cell wall and the cell protoplast becomes dehydrated
An environment external to the cell with lower solute concentration and higher water concentration
Indicate a role of pH in the control of microorganisms
Strong acid or alkali interfere with transport across cell membranes and block enzyme activity.
Describe two causes where distinct microorganisms are found in the body regions with a particular pH
Staphylococcus aureus is an example of a resident flora microbe found on the skin. The skin is pH 5. This microbe is able to grow at this pH. The acid pH is due to fatty acids released by the sebaceous glands on the skin surface. Bacteria in the genus Lactobacillus are normal flora in the vagina and ferment glycogen to lactic acid to help maintain the normal low pH of the body organ.
How can E. coli cause gastrointestinal and genitourinary tract infections?
This microbe can live in a pH of 8 which is common in the intestines. Many of the strains of Escherichia coli are toxin producers in the small intestine or can invade the mucosa of the small intestine. In both cases, enteritis is produced with diarrhea, cramps, and abdominal pain. Escherichia coli can also infect the genitourinary tract in women because of the close proximity of the anal opening and the genitourinary tract openings. This organism is sometimes transferred to and infects these tracts. This microbe can survive in the alkaline digestive tract and the acid genitourinary tract.
How can staph. aureus cause skin infections?
Staphylococcus aureus is normal flora of the skin and is able to live in the acid pH of the skin and infect hair follicles, produce boils and also is involved with pathogenic streptococci in impetigo.
Strong acids kill microbes by
Interfering with enzyme activity denaturing proteins, and blocking transport of materials into the cells.
Enteric pathogens survive the stomach acid because they are
Coated with food particles and protected against enzyme destruction
An intenstinal organisms known to produce pyogenic infections in other parts of the body is
Which kind of microbes present on skin will grow on the incubated trypticase soy agar plates?
Name some organisms commonly found as resident flora on the skin
Resident flora on the skin include members of the genus Corynebacterium and Staphylococcus epidermidis.
2 body mechanisms used to eliminate transient flora
Two mechanisms are the low pH of the skin which inhibits bacterial growth, and the enzyme action of lysozyme which destroys bacterial cell walls.
Examples of transient flora
S. aureus, gram-negative coliforms, clostridia and aerobic spore-forming bacilli that are commonly found in air, water and soil
Examples of resident flora
Diphtheroids such as Corynebacterium and Propionibacterium and staphylococci such as S. epidermidis
Diseases transmitted through milk
Two diseases transmitted through milk are tuberculosis and brucellosis.
Importance of pasteurization
Pasteurization eliminates the disease producing organisms that can be present in milk.
Thermoduric organisms are those bacteria that survive pasteurization treatment of milk.
Standards for Grade A milk after pasteurization*
The bacterial count for Grade A pasteurized milk cannot exceed 20,000 bacteria/mL and the coliforms are not to exceed 10/ml.
Nutrients found in milk which make it an excellent nutritional medium for growth of microorganisms
the carbohydrate lactose, the protein casein, lipids, calcium, phosphorous and vitamins.
Contamination of raw milk
Due to bacteria on the cow udder, utensils used in milking, and dust/dirt exposed during handling, raw milk becomes contaminated by pathogens
The neutral pH and various nutrients in milk make it a good environment for bacterial growth
72.5 C for 15 seconds
Eliminates most disease-producing organisms
Thermoduric organisms survive pasteurization
Streptococcus (S. lactis) and Lactobacillus
Ferment lactose to lactic acid (sours milk)
Some spore formers in the genus Bacillus
Digest milk proteins
Raw milk standards
Individual producer milk products can contain up to 100,000 bacteria/mL prior to commingling (mixing)
After commingling can contain up to 300,000 bacteria/mL
The Standard Plate Count
used to determine the approximate number of microorganisms in milk and milk products, and can be adapted to show the presence of specific organisms like coliforms
Fermentation is an oxidation-reduction biochemical pathway in which an organic compound such as pyruvate is changed to another organic compound such as lactate in the absence of any added electron acceptor.
Mixed acid fermentation vs. 2, 3 butanediol fermentation
Mixed acid fermentation produces a variety of acids such as acetic, lactic, and succinic acids which reduce the pH below 4.4 as well as ethanol, carbon dioxide, and hydrogen gas. Butanediol fermentation produces the neutral fermentation products acetoin and 2, 3 butandiol together with ethanol, carbon dioxide and hydrogen gas. Very little acid is produced. The pH is 6.2.
Why are the methyl red and Voges-Proskauer tests are useful in the identification of some enteric bacteria?
These two tests divide the Enterobacteriaceae into two groups. Bacteria in the genera Escherichia, Proteus, Salmonella, and Shigella produce a positive Methyl-Red Test and a negative Voges-Proskauer Test. Bacteria in the general Enterobacter, Klebsiella, and Serratia produce a negative Methyl-Red Test and a positive Voges-Proskauer Test.
Purpose of methyl-red in M-r test
The purpose of the methyl red indicator is to show the presence of mixed acids produced in the Methyl-Red Test. This indicator turns red at a pH of 4.4.
Oxidative vs. nonsaccharolytic metabolism of sugars
In oxidative metabolism, the Krebs Cycle and electron transport phosphorylation are used to aerobically break down sugar to carbon dioxide and water. Nonsaccharolytic metabolism means that the organisms do not use sugar at all.
The oxidation of the reduce coenzyme by fermentation is necessary for the anaerobic breakdown of sugar because it restores
the oxidized coenzyme necessary for conversion of 1,3 diphosphoglyceric acid to 3-phosphoglyceric acid.
-The process of energy release in cells that involves the anaerobic breakdown of glucose to pyruvic or lactic acid
-Two-stage pathway leading to the formation of ATP and pyruvic acid
Fermentation involves oxidation and reduction of organic compounds in a biochemical pathway in which no added electron acceptor is present
Some streptococci and lactobacilli carry out a homolactic fermentation and produce primarily lactic acid from pyruvic acid
Other streptococci and lactobacilli carry out a heterolactic fermentation and produce lactic acid, ethanol, and carbon dioxide
Include aerobic, non-spore-forming bacilli that are non-saccharolytic and do not use carbohydrate or metabolize carbohydrates in a strictly oxidizing fashion
Alpha vs. beta hemolysis
Alpha-hemolysis shows as a green halo around a colony on blood agar. This is due to partial destruction of the red blood cell membrane and hemoglobin. Beta-hemolysis shows as a clear halo around the colony. This is due to destruction of the red blood cell membrane and hemoglobin.
Why does S. aureus produce a yellow-colored halo around the colonies on mannitol-salt agar?
Staphylococcus aureus ferments the mannitol sugar in mannitol-salt agar to acids. The indicator, phenol red, changes from red to yellow and a yellow halo is seen around the colony. This medium contains 7.5% NaCl which inhibits most other organisms, thus making this agar highly selective for staphylococci.
Name a test that distinguishes b/t streptococci and staphylococci and list the results found.
Streptococci give a negative catalase test. Staphylococci give a positive catalase test. In a positive test the enzyme catalase converts the hydrogen peroxide produced in respiration into water and oxygen. profuse bubbling of oxygen is seen on the surface of the bacterial growth when the growth is flooded with hydrogen peroxide.
Indicate the importance of the coagulase test.
The coagulase test is the clinically definitive test for the identification of beta-hemolytic pathogenic Staphylococcus aureus.
Three viruses that cause upper respiratory infections are
rhinoviruses, adenoviruses, and respiratory syncythial viruses
3 bacterial organisms found in the throat are
diphtheroids, Staphylococcus aureus, Staphylococcus epidermidis, and Moraxella (Branhamella) catarrhalis
2 features of Klebsiella pneumoniae on blood agar are
nonhemolysis and mucoid glistening colonies on blood agar
3 tests that identify Staph. aureus are
fermentation of mannitol in mannitol-salt agar, reduction of tellurite to tellurium in tellurite agar, clotting of rabbit plasma by coagulase enzyme
A serious medical problem in children produced by Hemophilus influenza is
epiglottitis or enlargement of the epiglottis such that the airway is blocked and death by suffocation may result
Staph. epidermidis produces a _____ halo around the colonies growing on mannitol-salt agar.
Staphylococcus epidermidis does not ferment mannitol. It may grow on this medium and produce colonies surrounded by a red color to the medium
Clearing around colonies on DNA agar plates on addition of 1 N hydrochloric acid means _____ of the DNA
How is low pH is created on skin?
Sebaceous glands in the skin release sebum which contains complex lipids that can be broken down by lipase enzymes from Propionibacterium to fatty acids. These fatty acids lower the pH of skin to 5 which is inhibitory to many other gram-negative transient organisms.
Physiological effects that interfere with growth of transients on skin are _______
Intact skin is a barrier to transient organisms, outer layers of skin are periodically shed, sodium chloride (salt) is released from sweat glands and inhibits bacteria other than the staphylococci, acid pH of the skin (about pH 5), transient organisms must compete with resident organisms for space and nutrients, and the outer skin is somewhat dry.
S. aureus can cause what diseases?
Causes boils, carbuncles, impetigo, toxic shock syndrome and staphylococcal scalded skin syndrome
Hydrogen peroxide is added to bacteria which will cause bubbling if catalase enzyme is present but remains undisturbed if not present
Staphylococcal coagulase enzyme converts fibrinogen protein in plasma into insoluble fibrin threads, which results in the formation of a clot in the tube
Mannitol Salt agar (MSA)
Selective and Differential
Selective medium (7.5% NaCl) for halotolerant organisms like staphylococci
S. aureus ferments mannitol. The acid production causes the medium to turn yellow
Describe pyrimidine dimer formation during irradiation with UV light
During UV light irradiation of cells, adjacent pyrimidines on the same chain are joined together by a four carbon cyclobutane bond. This bond binds the two pyrimidines together. It weakens the attraction to the complementary bases in the opposite chain. Pyrimidine dimers interfere with DNA replication so that incorrect bases are inserted into the DNA and mutations occur. Accumulated thymine dimers are lethal to the cell.
Explain what is accomplished by photoreactivation
During photoreactivation, the photolyase enzyme, restores the pyrimidine dimer back to the original pyrimidine bases by breaking the cyclobutane bond.
T/F Wrapping irradiated plates with aluminum foil prevents any light induced radiation repair damage
T/F UV only irradiated plates show a lower survival than UV + photo-reactivation treated plates.
Formation of Pyrimidine Dimers
UV radiation in the range of 260 nm is strongly absorbed by purine and pyrimidine bases in DNA
When two pyrimidine bases are next to each other on the same DNA strand, and absorb UV radiation, they can form an unusual cyclobutane bond between their adjacent carbons
The thymine dimer is the most common pyrimidine dimer formed
Events in Photoreactivation (PHR)
Many microorganisms have repair mechanisms that can eliminate damage produced by UV light
In the presence of longer wavelength light (e.g., 500 nm), the organism's photolyase enzyme breaks cyclobutane bonds in the dimer and restores adjacent pyrimidines to their original shape
Bactericidal vs. bacteriostatic
A bactericidal agent kills microorganisms, while a bacteriostatic agents prevents multiplication of cells.
Describe the action of a cationic quarternary ammonium compound
Cationic quarternary ammonium compounds attach by their positive ends to the negatively charged phospholipids in the cell membrane and break apart the cell membrane causing leakage of materials, protein denaturation and cell death.
Describe the action of iodine-contaminating compounds
Iodine containing compounds attach to the amino acid tyrosine and denature proteins containing it in the cell.
Name an unusual feature of the action of alcohol as an antimicrobial agent
Absolute alcohol is not antimicrobial. Water is necessary for the protein denaturation effect of alcohols. 70% alcohol shows effectiveness against microorganisms.
Phenolic compounds damage bacterial cells by
denaturing proteins and damaging cell membranes so that nutrients leak out of the cells and they die.
Alcohols with a _____ molecular weight are more effective as antimicrobial agents that those with a _____ molecular weight
Phenol, cresols, orthophenylphenol, hexachlorophene and LysolTM
Break down the cell membrane of bacterial cells (nutrients leak out of the cells and the cells die)
Used to kill microorganisms on the skin prior to surgery
Iodine combines with the amino acid tyrosine inactivating and denaturing proteins containing it
Used in drinking water supplies and swimming pools as a disinfecting agent
Chlorine combines with water to form hypochlorous acid, which is a strong oxidizing agent
Dissolve lipids in cell membranes
Concentration and effectiveness:
Absolute alcohol is not effective as an antimicrobial agent since the presence of water is required for denaturation of proteins
Most effective concentrations are 70% to 80%
Quaternary Ammonium Compounds (quats)
Long-chain organic hydrophobic chains attached to a positively or negatively charged group
Positively charged quats are cationic compounds
Negatively charged quats are anionic compounds
Cationic compounds are more effective against bacteria than are the anionic compounds
Cationic compounds are also more effective against gram-positive than gram-negative bacteria
The positively charged cationic compound combines with the negatively charged phospholipids of the cell membrane
They break down the the cell membrane and denature the proteins
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