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184 terms

Lab Test #2

Lab Manual: Laboratory Applications in Microbiology by Barry Chess. Examples: 5,7,8,9,11,43,57, Appendix C.
STUDY
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Nosocomial
taking place or originating in a hospital
Aseptic
free of or using methods to keep free of pathological microorganisms
Sterile
free of or using methods to keep free of pathological microorganisms
Ubiquitous
being present everywhere at once
Pathogen
any disease-producing agent (especially a virus or bacterium or other microorganism)
Inoculate
to implant (a disease agent or antigen) into a person, animal, or plant to study
Turbid
(of especially liquids) clouded as with sediment
Potable
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?
Pathogenic
In the laboratory, what kind of unwanted microbe is capable of ruining an experiment?
Any
In positive pressure rooms, the air pressure is _____ than that found in the adjoining corridor.
Higher
in negative pressure rooms, the air pressure is ____ than that in the coridor.
Lower
Sputum
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.
Parent
Trismus
prolonged spasm of the jaw muscles
Aerobic
depending on free oxygen or air
Anaerobic
living or active in the absence of free oxygen
Sequelae
an abnormal condition resulting from a previous disease.
Colony
(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?
4
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?
24-48h
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?
Red
What color is a colony of Escherichia coli?
White
What color is a colony of Micrococcus luteus?
Yellow
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____.
Morphology
What shape is a cocci?
spherical
What shape is a bacilli?
rods
What shape is a spirilla?
spiral
What is the name for curved rods?
Vibrios
What are spirochetes?
flexible spiral bacteria
Pleomorphic
Pertaining to a variety of shapes or cell types.
A cocci dividing in one plane is called a
Diplococcus
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..
Staphylococcal
Rod-shaped bacteria divide only along the transverse plane. Bacilli arrangements consists of:
diplobacilli, streptobacilli, and palisades
Palisades
The characteristic arrangement of Corynebacterium cells resembling a row of fence posts and created by snapping.
What color are bacteria initially?
transparent
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?
violet
What is the final color in Gram negatives?
pink
Morphology
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
Arrangement
The way a bacteria grow:
diplo = pairs (e.g. diplococcus are round and are found in pairs)
strepto = chains
staph = clusters
single
Pleomorphism
(biology) the appearance of two or more distinctly different forms in the life cycle of some organisms
Contrast
The differences in values, colors, textures, shapes, and other elements within a presentation
Mordant
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
Endospore
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?
Heat
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?
Blue
Depletion of what causes endospores to form?
Carbon or nitrogen
What is the mordant in acid-fast staining?
Heat
What is the primary staining for endospores?
malachite green
What final color are Non-endospore formers?
Pink
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).
Sporangium
organ containing or producing spores
Bronchoscopy
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
Prophylactic
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.
Acid-fast
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?
Non-acid-fast
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?
Flagella
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
Heterotrophs
microbes that obtain carbon from organic compounds such as carbohydrates and proteins
Autotrophs
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
chemo-
energy is derived through the breakdown of chemical substrates
photo-
light is used to provide energy through photosynthesis
Chemoorganotrophs
derive energy from the breakdown of organic molecules by fermentation or respiration (most bacteria and humans fall in this category)
Chemolithotrophs
rely on inorganic ions as an energy source, oxidizing inorganic substrates such as sulfur or iron to obtain energy
Photoautotrophs
use photosynthesis pigments to convert sunlight into chemical energy through the process of photosynthesis
Photoheterotrophs
use light as a source of energy, but carbon is obtained from the breaking down of organic molecules such as glutamate
Nitrogen
is essential for the synthesis of amino acids, nucleotides, and a few other cellular constituents
Minerals commonly required for bacterial metabolism include:
sodium, potassium, calcium, magnesium, manganese, iron, zinc, copper cobalt, and phosphorous
Two genera that is unable to synthesize all the vitamins on their own:
Streptococcus
Lactobacillus

(so they need vitamins added in media in able to grow)
Growth factors
complex organic compounds that are required for the growth of some fastidious organisms
Enriched media
blood or serum is added to complete media to ensure the growth of certain bacteria
Vitamins
complex organic molecules that serve as cofactors in many enzyme-catalyzed reactions
Bacterial cells consist of __ % of water, or more.
70%
What kind of water is used in preparing media?
distilled or deionized water as tap water often contains ions such as calcium or magnesium that can interfere with bacterial growth
Selective medium
contains one or more ingredients that inhibits the growth of most bacteria, allowing only a single group to grow
Conditions used for selective mediums:
dyes, salts, and antibiotics
Differential medium
allows all species to grow, but the inclusion of a specific ingredient in the media results in some bacteria appearing different than others
Mannitol salt agar has which type of media properties?
Both selective and differential
Liquid media
referred to as broths, milks, or infusions. are, at their most basic, simply nutrients dissolved in water
Solid media
has the same formula as the liquid media except that a solidifying agent is added so that the media is solid at temperatures used to incubate bacteria; eg. agar
Agar
a polysaccharide isolated from the red algae Gelidium
Agar melts at what temperature?
100 degree Celsius
Agar solidify at what temperature?
42 degree Celsius
Bacteria can be inoculated into melted agar at what temperature without killing the cells?
50 degree Celsius
How many % of agar is considered a semi-solid?
0.4%
How many % of agar is considered to be solid?
1-2%
Motility is tested in which media state?
Semi-solid
Why is agar used over gelatin?
-gelatin liquidfy at room temperature
-very few bacteria can utilize agar as a nutrient
What two things can be used to check for pH?
pH meter
pH paper
What do you use if the pH in a media is too low?
NaOH: 1N and 0.1 N solutions, to adjust the pH upward
What do you use if the pH in a media is too high?
1 N and 0.1 N HCl to adjust the pH downward
Once media is prepared, it must be autoclaved immediately because:
bacteria in the water, on the inside walls of the tube, and in the dehydrated medium will soon begin to grow, destroying the medium
Caps must be loose in tubes of media before autoclaving to allow what?
steam to escape
Sterilization occurs at what temperature?
121 degree Celsius, or 15 psi(pounds per square inch)
Small loads in the autoclave takes how long?
10-15 minutes
Full autoclave may require how long?
30 minutes
How long does it take the agar to solidify after audoclaving?
30-60 minutes
Broths, deeps, and other similar media should be allowed to cool to _____ temperature, then stored in the _________.
room; refrigerator

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