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Objective of oil immersion lens

100X

Resolving power becomes _____ as magnification of the objective lens _______

Greater, increases

The light microscope can distinguish two points that are at least _____ um apart

0.2

What is used to sterilize heat sensitive liquids?

Filtration

The streak plate technique uses spatial dilution (Increases the space b/t organisms) to create

pure colonies

The component of MacConkey agar that inhibits Gram-pos. growth is

bile salts

Asepsis means

lacking viable microorganisms

______ means a specimen in focus under the low power objectives should be nearly in focus under high power objectives as well

Parfocal

What is a fomite?

Non-living object. I.e. door knob

Pure culture is

progeny from one isolated cell

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.

Define a simple stain

A simple stain involves staining bacteria with a single dye.

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.

Basic dyes consist of

positively charged color group and a negative anion.

An example of a basic dye is

crystal violet, safranin

Rinsing the slide thoroughly serves to

removes excess dye and prevents crystal formation.

Stained Bacillus mycoides apear as ______ when viewed in the microscope

rods in chains

An acidic dye has a _______ charged color group

Negatively

Heat fixation kills cells while _____ distortion of the cells

minimizing

_______ dyes are used to stain common bacteria like Staphylococcus

Basic

A simple stain shows

cell morphology (shape) and arrangement and does not distinguish between different types of bacteria (like G+ and G-)

Staining provides contrast between _____ and the light field of the microscope

the bacteria

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

The amount of light sent to the stage opening is regulated by the

Iris diaphragm

The lens that one looks through is called the

ocular lens

Focusing under high power is done with the

fine adjustment

As magnification ______, resolving power _____

increases, increases

As magnification ________, working distance _____

increases, decreases

The stage or objective is moved the greatest distance by the

course adjustment

_____ is the ability of a lens in a light microscope to gather light

Numerical aperture

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.

Environmental surfaces include

non-living surfaces

Infectious diseases can be transmitted by

fomites, aerosols, and by contaminated food and water

The majority of microorganisms are

non-pathogenic, and do not cause disease

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.

Nutrient broth contains distilled water and _____

Beef extract (and peptone)

Sterile =

free from living microorganisms

Growth is detected in broth by ______

turbidity (cloudiness in medium)

Autoclave or steam sterilization

Uses steam under pressure and is sterilization of culture media, reagents, and laboratory tools

Dry Heat

Uses an oven to generate hot dry air
Sterilization of glassware and moisture sensitive preparations

Filter sterilization

Uses a membrane to trap microorganisms
Sterilization of heat sensitive liquids

Flame sterilization

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.

Phenylethyl alcohol inhibits ______

gram-negative bacteria.

Differential media

the growth in unique fashions of different kinds of microorganisms.

Lenticulate colonies are found _____ the surface

below

Staph. aureus forms ____ colonies on PEAB

white

Bacillus subtilis forms ____ colonies on nutrient agar

cream

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

MacConkey agar

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

Microorganisms occur

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

Capsule =

is a gelatinous covering around the outside of a cell that adheres tightly to the cell surface.

Composition of a capsule

composed of polysaccharides.

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.

The negative capsule stain uses the chemical

india ink

violet staining of the cell is due to

crystal violet

capsules interfere with a process in neutrophils called

phagocytosis

complement _______ encapsulated cells

doesn't affect

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

Quelling test

Capsular swelling in the presence of specific antibody
Valuable laboratory test for the sero-identification of specific encapsulated bacteria

What kind of dye is used for a simple stain of a bacterial cell?

Basic

The color of an endospore in the Gram stain is

clear

What is the de-colorizer in the gram stain?

95 % ethyl alcohol

What genera is acid-fast?

Mycobacterium

Endospore(s)

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

Two chemicals that contribute to endospore resistance are

calcium and dipicolinic acid

In a negative stain, the purpose of India ink is

to create a color contrast b/t the capsule and background light

Primary dye

The primary dye is the first dye used in a differential stain such as the Gram stain.

Mordant

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.

Decolorizing agent

The decolorizing agent removes the primary dye from a cell so that the cell is colorless

Counterstain

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.

Gram + cells are more sensitive to agents such as

detergents, phenol, and penicillin

Old gram positive cell cultures become ____

gram variable

Klebsiella pneumonia is gram

positive

Pseudomonas aeruginosa appear as ______ cells in the Gram procedure

rod-shaped

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

The acid-fast state depends on what property of a cell?

Waxy lipid (mycolic acid) in cell walls

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.

The primary dye in the acid fast stain procedure is

carbol fuchsin

Decolorizing agent in acid fast is

acid-alcohol

Acid fast cells stain

pink

Counterstain in acid fast is

methylene blue

Endospore

An endospore is an oval, metabolically inactive structure that is formed within the bacterial vegetative cell.

Sporangium

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.

What significant medical probs are posed by spores?

Gas gangrene, anthrax, botulism

Gram _____ bacilli form spores

positive

They dye used to stain the endospore is

malachite green

Counterstain for endospores is

safranin

Endospores are resistant to

dry freezing, UV light, high heat, chemicals, etc.

E. coli _____ produce endospores

doesn't

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

Brownian movement

Random back and forth movement of a cell

Staphylococcus aureus uses proline to

build up within the cell and raise the osmotic pressure

Gram stain does not identify what characteristic?

motility

Methods for observing motility

Hanging drop preparation, Stab inoculation into motility deeps

Which temperature grown classification grows best at 37 C but can also grow at 0 C?

Psychrotrophs

The greatest rate of cell growth for a thermophile is

optimal temperature

A unique characteristic of extreme halophiles is that they

require a high salt concentration

Cells in a hypertonic environment causes

Water loss and plasmolysis

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.

Positive chemotaxis occurs in presence of

flagellated bacteria

Micrococcus luteus is ____

non-motile

True motility vs. Brownian

True motility requires energy expenditure by the cell, Brownian motion results from kinetic energy of molecules around the cell

Chemotaxis

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.

Endospores are commonly found in the groupings

Bacillus or clostridium

Erythromycin primarily affects cells that are Gram

positive

Pigments produced by bacteria can color

the medium or the colonies or both

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

Psycrophile

Psychrophiles grow at -5 C to 20 C. Pseudomonas fluorescens is a psychrophile

Mesophile

Mesophiles grow at 20 C to 45 C. Escherichia coli and Staphylococcus aureus are mesophiles

Thermophile

Thermophiles grow at 45 C to 60 C. Bacillus stereothermophilus is a thermophile.

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.

Food spoilage at somewhat elevated temps may be due to

thermophiles

At the max temp for growth, reproduction is reduced due to

protein denaturation, loss of enzyme function, and cell death

Growth in microorganisms means

increase in cell number

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.

Bacterial cells immersed in a hypotonic environment tend to

gain water

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