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Lab Manual: Laboratory Applications in Microbiology by Barry Chess. Examples: 5,7,8,9,11,43,57, Appendix C.


taking place or originating in a hospital


free of or using methods to keep free of pathological microorganisms


free of or using methods to keep free of pathological microorganisms


being present everywhere at once


any disease-producing agent (especially a virus or bacterium or other microorganism)


to implant (a disease agent or antigen) into a person, animal, or plant to study


(of especially liquids) clouded as with sediment


any liquid suitable for drinking

How would you recognize microbial growth in a liquid media? What about a solid media?

In liquid: medium will have precipitation in it.
In solid: colonies will form

What is a colony? What is the relationship between a cell and a colony?

A cell is a unit of a colony. A colony has millions of cells. A colony can be seen by the naked eye, but a cell needs a microscope.

What additional information does a solid culture provide compared to a liquid culture?

A solid culture is a a differential medium in which the colors differentiate the different microbes. The microbes look the same in a liquid medium.

What are some characteristics of bacterial and fungal colonies that can be used to differentiate between the two?

Bacterial colonies looks like little dots and has color. Fungal colonies are furry/fuzzy with hyphae

In hospitals, we are primarily concered with the spread of what kind of microbes?


In the laboratory, what kind of unwanted microbe is capable of ruining an experiment?


In positive pressure rooms, the air pressure is _____ than that found in the adjoining corridor.


in negative pressure rooms, the air pressure is ____ than that in the coridor.



matter, as saliva mixed with mucus or pus, expectorated from the lungs and respiratory passages.

Explain what is meant by a false positive result. How does this differ from a false negative result?

A test result which indicates that an individual is affected when he or she is actually unaffected; ie, a positive test result in a truly unaffected individual. A false negative is tested negative, but they do have the disease.

The pulmonary disease tuberculosis (TB) is caused by what bacterium?

Mycobacterium tuberculosis

What will happen if a hot inoculating tool is plunged into a media containing bacteria?

An aerosol may be created, releasing bacteria into the air.

When bacterial growth is being removed from a liquid culture, which inoculating tool is used?

A loop

When bacterial growth is being removed from a solid culture(slant or plate), which inoculating tool is used?

A needle

Tilting the tube when inoculating prevents what?

Airborne contamination

In the "TB" case, how did contamination most likely occur?

Not using aseptic techniques, poor handling of a the test culture which allowed cross contamination.

Pure culture

single species is isolated from other species in the population (opposite of mixed culture)

Mixed culture contains how two ____ cells.



prolonged spasm of the jaw muscles


depending on free oxygen or air


living or active in the absence of free oxygen


an abnormal condition resulting from a previous disease.


(microbiology) a group of organisms grown from a single parent cell

What is the difference between a pure culture and a mixed culture?

A pure culture contains one cell and a mixed culture contains many cells.

How can a pure or mixed cutlure become contaminated? How can this be prevented?

They become contaminated when another unwanted microbe goes into the medium. This can be prevented by aseptic techniques.

What is the streak-plate method?

This method is the most economical in terms of time and materials, requiring just a few minutes and only a single plate of media.

How many quadrants does a streak plate have?


Agar must be cooled to what temperature?

50 degree Celcius

How long do you have to incubate the plate in an inverted position at 25 degree Celcius?


What is the loop dilution method?

This method consumes more time and materials than does a streak plate, but the results produced, even by the beginning student, are generally quite good.

What is the spread-plate method?

This method gives consistently reliable results when bacterial samples are dilute or if the medium being inoculated is highly selective.

What is the importance of generating isolated bacterial colonies?

To be sure that only one cell is being examined and that it is a pure culture.

What is a subculture?

to cultivate (a bacterial strain) again on a new medium.

What is the purpose of subcultiring bacterial isolates?

allows easier study and organization of bacterial isolates

What color is a colony of Seratia marcescens appear?


What color is a colony of Escherichia coli?


What color is a colony of Micrococcus luteus?


What bacterium causes neonatal tetanus and where is it found?

Clostridium tetani (found in soil and animal feces)

Is Clostridium tetani an aerobe or anaerobe?

Anaerobe, only grow in the absence of oxygen..

Agar solidifies in what temperature?

Room temperature

Cells of a given species all generally have the same shape, or____.


What shape is a cocci?


What shape is a bacilli?


What shape is a spirilla?


What is the name for curved rods?


What are spirochetes?

flexible spiral bacteria


Pertaining to a variety of shapes or cell types.

A cocci dividing in one plane is called a


A diplococcus continues to divide into...

Streptococcus (variable number of cells)

A cocci divinding in two perpendicular planes is called a

Tetra (4 cells)

A tetra continues to divide into..

Sarcina (packet of 8-64 cells)

A cocci dividing in irregular cluster of cells is called..


Rod-shaped bacteria divide only along the transverse plane. Bacilli arrangements consists of:

diplobacilli, streptobacilli, and palisades


The characteristic arrangement of Corynebacterium cells resembling a row of fence posts and created by snapping.

What color are bacteria initially?


Negative staining stains what?

The background, which is dark and the cell is light.

Positive staining (simple staining) stains what?

The bacterial cell so that it appears as a darkly colored object against a light background.

Two types of negative stains:

Nigrosine, and India ink.

Common basic (positively charged) stains in the microbiology laboratory:

methylene blue, crystal violet, malachite green, and safranin (which is pink)

Which staining provides little information?

Negative staining

What is one advantage of negative staining?

Has the most accurate determination of the size of a bacterial cell because it doesn't require heating(like positive staining), which shrinks the cell.

Gram stain is considered what kind of staining?

Differential staining

What is the order of Gram staining?

crystal violet(primary stain), Iodine(mordant), ethyl alcohol(decolorizer), safranin(counterstain

What is the final color in Gram positives?


What is the final color in Gram negatives?



1. scientific study of the forms and structures of plants and animals 2. form and structure of an organism or any of its parts
cocci = spheres
bacilli = rods
vibrio = bent rods
spirilla/spirochetes = spiral shaped


The way a bacteria grow:
diplo = pairs (e.g. diplococcus are round and are found in pairs)
strepto = chains
staph = clusters


(biology) the appearance of two or more distinctly different forms in the life cycle of some organisms


The differences in values, colors, textures, shapes, and other elements within a presentation


having the property of fixing colors, as in dyeing

Cellular morphology of Salmonella typhimurium.

Rod-shaped, Gram-negative

Cellular morphology of Staphylococcus aureus.

Cocci-spherical, Gram-positive

Cellular morphology of Listeria monocytogenes.

Rod-shaped, Gram-positive

Cellular morphology of Bacillus cereus.

Rod-shaped, Gram-positive

What two staining techniques are appropriate for determining the shape and arrangement of a bacterial species?

Differential and Gram staining

What stains can be used for negative staining, and why can these same stains not be used for simple staining?

Negative staining needs negative charged acidic dyes(nigrosine or India ink) and positive staining needs positive charged dyes.

What is the difference between a simple stain and a differential stain?

Simple stain is a positive stain which stains a pure culture. Differential stain like the Gram stain, can be used to to a mixed culture.

Cellular morphology of Listeria innocua.

Rod-Shaped, Gram-positive

Cellular morphology of Bacillus magaterium.

Rod-shaped, Gram-positive

Based strictly on cellular morphology, which bacterial species is most easily differentiated from the other? Why?

Staphylococcus aureus & Salmonella typimurium
They have different shapes and structures.
Gram-positives and Gram-negatives can be distinguished by color.

Time needed for primary stain:

30 seconds

Time needed for mordant:

60 seconds

Time needed for decolorizer:

8-15 seconds

Time needed for counterstain:

60 seconds


A small, dormant, resistant derivative of a bacterial cell that germinates under favorable growth conditions into a vegetative cell.

What two bacteria are typical spore formers?

Bacilus, Clostridium, and Sporosarcina

What is the use of acid-fast stains?

To detect members of the genus Mycobacterium, such as the pathogens M. tuberculosis and M. leprae which causes leprosy. Other bacterial species in the genus Nocardia and protozoan parasites (Cryptosporidium and Isospora). Only a few are acid-fast organisms.

Acid-fast bacteria contains what in their cell walls?

Mycolic acid - a waxy material which prevents stains from penetrating

What is used to soften the mycolic acid?


What is the primary stain for acid-fast?

carbol fuschin (deep red)

Cells that are non-acid fast are easily decolorized by:

acid - alcohol

Counterstain for acid-fast stains:

methylene blue

What is the final color of non-acid-fast bacterias?


Depletion of what causes endospores to form?

Carbon or nitrogen

What is the mordant in acid-fast staining?


What is the primary staining for endospores?

malachite green

What final color are Non-endospore formers?


What are the steps of Endospore staining?

malachite green(primary stain), heat(mordant), water(removes green dye from vegatative cells but not from endospores), safranin(counterstain vegatative cells and sporangium).


organ containing or producing spores


the visual examination of the bronchi using a bronchoscope

Etiological agent

The organism (species or strain) responsible for producing a disease

Bilateral infiltrates

like an x-ray


defending or protecting from disease or infection, as a drug, such as antibiotics

Differential stain

stains to react differently with different types of bacteria, used to distinguish bacterial types. Gram stain, acid-fast stain

Two diseases caused by acid-fast bacteria and the species related:

TB: Mycobacterium tuberculosis
Leprosy: Mycobacterium leprae

Four bacterial pathogens that form endospores and the disease associated with each:

Bacillus anthracis : Anthrax
Clostridium botulinum : paralysis muscles, food poisoning
Clostridium tetani : lockjaw
Clostridium perfringens : gas gangrene(blockage of blood supply)

Cellular morphology and arrangement of Mycobacterium smegmatis.

Bacillus shape, acid-fast

Cellular morphology and arrangement of Staphylococcus aureus.

Cocci-clusters, non-acid-fast

What causes acid-fast cells to clump together?

Mycolic acid - a waxy material that prevents stains from penetrating

Why is anthrax also known as Woolsorter's disease?

Because anthrax initially started when human ingest spores from wool pelts.

What is secondary contamination?

radioactive contamination that has been transferred from one location to another, such as from hands to face or from a contaminated tool or from contaminated protective gear to one's skin; it can also refer to transfer from a contaminated individual to a second individual. Secondary contamination is often referred to as cross-contamination.


not easily decolorized by acid solutions; pertains to micro-organisms (especially the tubercle bacillus that causes tuberculosis), organisms that demonstrate a characteristic of having a high lipid content in their cell wall that resists drying, acids and various germicides?


don't have mycolic acid so they lose color during decolorization. to be visualized, must be counterstained with methylene blue

What makes staining endospores a challenge?

the protective nature of the endospore prevents the penetration of dye

Motile bacteria display...

chemotaxis, a complex movement of the cell toward nutritients or away from harmful substances

What is the major motility structures found in bacteria?


What two methods are used to directly determine motility?

Wet mount
Hanging drop slide

Wet mount

a procedure in which a small amount of liquid culture is placed on a microscope slide and covered with a cover glass

Problems from using a wet mount:

-tendency to dry out
-current movements that may mimic motility
-potential for contamination of instruments and self with pathogenic microbes

Hanging drop slide prevents these problems

Hanging drop slide

a cover glass is prepared with a small dab of petroleum jelly (Vaseline) on each corner and a single loopful of a liquid culture in the center

Slide of a hanging drop slide has:

a depression in the middle

Brownian motion

movement due to water currents, bombardment of cells by surrounding molecules, primarily water; causes cell to jiggle or shake

Why is speed imporatant in these techniques to find motility?

time and evaporation of the media will reduce bacterial motility while condensation forming on a hanging drop slide can reduce the clarity of the image

Type of movement found in motile bacteria:

true directional movement

Type of movement found in nonmotile bacterias:

Brownian motion

Why is understanding motility in a bacteria useful?

used as an aid in the identification of an isolate(to single out)

True motility

movement in a single direction that is several times the length of the bacterium

Is Micrococcus luteus motile?

No, it is non-motile

Is Proteus vulgaris motile?

Yes, it is motile

All media can be classified as either:

complex or defined

Complex media:

contains one or more ingredients that are not precisely known, often an extract of animals, plants, or yeast (most media in the lab); eg.trypticase soy agar and nutrient broth

Defined media:

a precisely known chemical composition, with each ingredient weighed and added to the media during preparation; eg. minimal agar

Bacterium must have what to grow?

carbon, energy, nitrogen, minerals, vitamins, growth factors, and water

Carbon sources

Carbon forms the backbone of all organics molecules found in the bacterial cell

Organic molecules found in bacterial cell

proteins, carbohydrates, nucleic acids, and lipids


microbes that obtain carbon from organic compounds such as carbohydrates and proteins


microbes that are able to use carbon dioxide as its sole source of carbon

Energy sources

is required to assemble the raw materials found in the media and the biomolecues needed for continued cell growth


energy is derived through the breakdown of chemical substrates


light is used to provide energy through photosynthesis


derive energy from the breakdown of organic molecules by fermentation or respiration (most bacteria and humans fall in this category)

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