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Biology Laboratory Final Exam


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organisms that are the most widely distributed, simplest in morphology, smallest in size, most difficult to classify, hardest to identify
6 characteristics of bacteria
1) Ubiquitous
2) Simple morphology (shape)
3) Small size (microscopic)
4) difficult to classify and identify
5) prokaryotic, belongs to kindgom Monera
6) Generally non-photosynthetic
Proper labeling of a plate
1) TA name
2) What is incubated?
3) Date
4) Media name
5) Initials
any object, surface, or even a body part
Most organisms are _____________ and do not reside on a particular host
Free living
any area where a microbe resides and serves as a potential source of infection
Most microorganisms are __________________; they are normally harmless but can cause infection when given the chance
Opportunistic pathogens
- unpigmented, motile organisms
- belongs to Kingdom Protista
- eukaryotic
- single-cell transparent organisms, cell membrane, no cell wall
- in lab, paramecium are most common to be seen
- greenish or golden brown
- belongs to Kingtom Protista and includes all photosynthetic eukaryotic organisms
- no tissue differentiation
- distinct, visible nuclei and chloroplasts
- unicellular or colonial
- blue-green
- belongs to Kingdom Monera
- prokaryotic type of nucleus
- phototrophic bacteria: bacteriochlorophyll
allows one to see cellular structure of bacteria
Smears should do this
- withstand washings
- not be too thick
- not result in excessive distortion of organism
aggregate of millions of cells that come from one cell
Purpose of heat-fixing
- to kill bacteria
- to fix them to the slide
- to coagulate cytoplasmic proteins to make cells more visible
using a single stain to color bacteria due to their transparent nature
Simple staining
Common dyes
methylene blue, crystal violet, basic fuchsin
Why do common dyes work?
They have chromophores with positive charges and bacteria are negatively charged
Two types of dyes
Basic and Acidic
- have positive (+) charge
- ex. Methylene blue
- can penetrate bacteria because of the attraction between charges
Basic dyes
- have negative charge
- ex. Eosin, Nigrosin
- can't penetrate bacteria because of repulsion between the charges
- stains background
Acidic dyes
- stains the background of the slide and not the organism
- useful for determining
cell shape and size
- these 2 cultures used in this test:
- no heat fixing means to cell distortion
Negative staining; K.pneumoniae, S.epidermidis
2 staining techniques used for capsular staining
Negative and Simple
Capsular staining technique uses ________ to color slide background and uses ___________ to color cell proper of the bacteria
Congo Red; Maneval's stain
The _________ is the defining part of whether the bacteria is Gram-negative or Gram-positive. The _____________ is based on the composition/appearance of [it]
Cell wall; Differentiation
The major element of the cell wall in bacteria is ___________
For Gram-positive bacteria, the cell wall is mostly __% peptidoglycan; For Gram-positive bacteria, the cell wall is only ___% peptidoglycan
~90%; ~10%
- most Gram-positive walls have _______________ attached to them, which are partly responsible for the negative charge of the cell
teichoic acids
Gram-negative bacteria have a second layer besides the peptidoglycan layer called the _________________ because it is made of lipids and a variety of sugars
Outer membrane (Lipopolysaccharide layer)
In both types of cell wall, _________ forms an insoluble compound with _____________. A thick peptidoglycan layer prevents ______________ because the alcohol causes dehydration and shrinks the cell wall, so the dye becomes trapped
Iodine; Crystal violet; Decolorization
This is a differential stain used to detect the presence of mycolic acid
- rod-shaped, Gram-positive bacteria which like to grow at ~30 degrees C
- have unique waxy lipid layer called _____________ which makes the colonies look waxy and crusty
- resistant to dehydration
Mycobacterium; Mycolic acid
food that is used for culturing bacteria and other microorganisms
Three consistencies of Media
1) Liquid
2) Solid
3) Semisolid
A good solidifying agent:
1) is not utilized by the microorganisms
2) doesn't inhibit bacterial growth
3) Doesn't liquify at room temperature
This is sometimes used as a solidifying agent, but it is hydrolyzed by many organisms and liquifies at room temp
Two good choices for solidifying agents
Agar and silica gel
chemical compounds that can be broken down into or used to make monomers
nutrients required in large amounts: ex. C, N, P, S, K, Mg, Ca, Na, Fe
nutrients required in small amounts: ex. metals
7 Nutritional requirements for media preparation:
1) water
2) carbon
3) energy
4) nitrogen
5) minerals
6) growth factors
7) pH
Media that has been synthesized; has exact composition; compounds used are highly purified and defined
Synthetic or defined Media
Media where exact composition not known; utilizes compounds that include all nutrients but aren't defined
Non-synthetic or complex Media
4 Types of Special Media
1) Selective
2) Differential
3) Minimal
4) Enriched
Media that contains the basic requirements for the bacteria to grow such as salts, a carbon source, and nitrogen
Minimal Media
media that contains all basic requirements but adds amino acids, vitamins, and minerals
Enriched Media
media that allows only certain organisms to grow; selection based on absence of certain nutrients or presence of inhibitory substance
Selective Media
media that contains substances which change the appearance of one species allowing for differentiation; ex. EMB-Eosin Methylene Blue
Differential Media
an acidic dye which causes coliforms (bacteria found in the intestine) to turn a metallic green color
the killing of bacteria or endospores found in media or glassware; can be perfomed using an _____________
Sterilization; Autoclave
Minimum requirements for Sterilization
For 1 liter of Media, 15 minutes at 121.6 degrees C (250 degrees F) and 15 psi (pounds per square inch
2 methods for obtaining pure culture:
Streak plate and Pour plate
Streak plate has 4 techniques:
1) Quadrant streak
2) Radiant streak
3) 'T' streak
4) Continuous streak
Methods uses plates and agar tubes to isolate loopfuls of bacteria; requires less skill and practice but a lot of media and supplies
Pour plate
When grown at 25-30 degrees C, this organism produces a red pigment called ____________. When grown at higher temperatures, it appears _________ on the media. It is found all over the environment. What is this organism?
Prodigiosin; white; Serratia Marcescens
This organism has endotoxins which cause disease like food poisoning. It is found all over the environment, and is cream colored. What is this organism?
Escherichia coli
This organism is a common laboratory contaminant, is found all over environment, and yellow colored due to carotenoid pigments. What is this organism?
Micrococcus luteus
This Agar is used to isolate and differentiate pathogenic organisms like Staphylococcus aureus. It is a selective and differential media
Mannitol Salt Agar (MSA)
This Agar is used to isolate Streptococci and Staphylococci from mixed cultures. It is a selective media. It selects for Gram positive bacteria and inhibits the growth of Gram negative bacteria
Phenyl Ethyl Alcohol (PEA)
This agar used to isolate fecal coliforms. It is a selective and differential media. Its sugars provide fermentable substrates that promote the growth of fecal coliforms
Eosin Methylene Blue (EMB)
This agar used to select and differentiate a member of Enterobacteriaceae; a selective and differential media; contains lactose, bile salts, neutral red, and crystal violet
MacConkey Agar
Consists of yeasts and molds; belong to Kingdom Myceteae; Mycology is the study of this
Type of mold that has mass of intermeshed hyphae; what is seen microscopically
microscopic filaments that may or may not have walls
Has pseudohyphae which are multicellular structures produced through budding; has spores, which are smaller in size than parent cell and produced by budding
viruses that infect bacteria; _________ that paratisize E.Coli are referred to as ______________
phages; coliphages
2 types of viruses
Lytic and Lysogenic
type of virus considered virulent; enters cell, takes over, replicates, then lyses cell for release
type of virus considered temperate; enters cell, integrates into DNA of host; cell replicates normally then eventually viruses replicate and lyse cell to be released
Viruses are observed with __________ microscope; they're quantified by their effect on host cells so you count the ___________
electron; VIUs (virus infection unit)
3 steps to isolate bacteriophage from sewage
1) Enrichment (performed in rows)
2) Filtration (rows)
3) Separation (pairs)
This type of isolation increases number of phage in raw sewage; performed by combining ___________ (source of phage), __________ (host of phage), and __________ (DSPB). The DSPB increases the host #, which increases the source #.
Enrichment; sewage; E.Coli; Deca Strength Phage Broth
This type of isolation separates the E.Coli from the phage; uses a ___________ filter which allows the virus to pass but not bacteria; sample must be ______________ to rid all cellular debris so membrane isn't clogged; use ______ plates, 3 per filtration
Filtration; 0.2 nanometer pore size; centrifuged; EMB (Eosin Methylene Blue)
This type of isolation is used to find evidence of phage and to find the concentration of phage in original filtrate
an organism's ability, or lack thereof, to grow in the presence of oxygen
5 Oxygen classes of bacteria:
1) requires oxygen, uses it as final electron acceptor
2) grow in the presence or absence of oxygen, but prefer oxygen
3) cannot grow in presence of oxygen
4) requires free oxygen, but only in limited amo
1) Aerobe
2) Facultative Anaerobe
3) Anaerobe
4) Microaerophiles
5) Capnophiles
4 Types of Media for determining Oxygen requirements:
1) TSA deep
2) TSA shake
3) Fluid Thioglycollate Medium (FTM)
4) TSA plate
solid agar; more oxygen at the top of the agar to less or no oxygen at the bottom of the tube
TSA deep
Agar kept in liquid state by placing tube in warm waterbath. After inoculation, tube gently rolled between palms to disperse bacteria, then is allowed to solidify. As it solidifies, oxygen gradient is created
TSA shake
basic medium, oxygen gradient formed in media during cooling; contains ___________ and ___________, reducing agents that reduce oxygen present in media to water; has __________, a dye used as an oxygen indicator (pink, oxygen present) (colorless, oxygen
Fluid Thioglycollate Medium (FTM); Thioglycollate, L-cystine; Resazurin; Agar, gelatin
gas-generator envelope used for providing anaerobic environment; has sachet of __________ which acts as catalyst
GasPak apparatus; palladium
____________ tests for cytochrome, part of electron transport chain. It has positive (___________) and negative (____________) control; Positive result: _____________ color change within 15 seconds. Negative result: ________________ within 15 seconds
Oxidative Test; P.aeruginosa; E.Coli; Dark blue (purple); no color change
Sugar Fermentation Tubes test for fermentation of 4 sugars:
1) Glucose
2) Lactose
3) Mannitol
4) Sucrose
S.F.T's reagent: pH indicator is _____________, detects acid production among fermentation; pH of media will drop as acid is produced; (_______ for pH}7 and __________ for pH{7)
Phenol Red; Red; Yellow
Results for S.F.T's: If positive, ________ color change and ___________ in tube for gas production. If negative, _________ color change and ___________ in tube for no gas production
Yellow; Bubble; Red; No Bubble
_______________tests for presence of nitrate and presence of nitratase. During test, add 15 drops of each reagent to test and determine color change, if none, add _________. Results: positive if ____ color change or ____ color change once ______ is added
Nitrase Test; Zinc;Red, no, zinc; no, zinc
___________ looks for presence of emzyme catalase which breaks down toxic oxygen compounds into water and oxygen. Reagent: _______________. Performed using TSA slant. Positive result: ___________. Negative result: ___________.
Catalase Test; Hydrogen peroxide; Bubbles; No Bubbles
___________ (MR Test) tests for production of acids upon glucose fermentation. Reagent: ___________. pH indicator: red for pH _______, yellow for pH ________. Positive: ______. Negative: _________
Methyl Red; Methyl Red; {5; }5; Red; Yellow
___________ (VP Test) tests for production of alcohol; Look for color change: positive, __________; negative, _________
Voges-Proskauer; Barritt's reagents; Red; Yellow
______________ tests for unknown bacteria and utilization of citrate. Positive: ___________ color. Negative: ___________ color.
Simmons Citrate Test; Prussian Blue; Green
______________ tests for unknown bacteria and presence of phenylalanase. Positive: ____________ color. Negative: ___________ color.
Phenylalanine Deanimation Test; Deep Green; Yellow
_________ tests for unknown bacteria and presence of H2S production; semi-solid media; Positive: __________, cloudy tube, spreads throughout, dark precipitate. Negative: growth only on _____________, no dark precipitate
SIM Media Deep
___________ tests for unknown bacteria and presence of H2S production, fermentation of glucose, gas production. Positive: ____________, cracks in media, dark precipitate; Glucose if _____ color, ______ at bottom of tube. Negative: no cracks in media, no
Kilgar's Iron Agar (KIA); gas production; red, yellow; dark precipitate, red, red
Hydrolysis Test, looks for urease: reagent Phenol Red; Positive: _________, Negative: _______
Urea Hydrolysis; Red, Yellow
Hydrolysis Test, looks for tryptophanase: Positive if ________ at top, negative if _______ at top
Tryptone Hydrolysis; red ring; no red ring
Hydrolysis Test, looks for lipases; media is _________; positive _____________; negative _____________.
Fat Hydrolysis (SBA); Spirit Blue Agar; Dark Precipitate; no dark precipitate
Hydrolysis Test, looks for presence of caseinase; Positive, ________ around growth; negative, _________
Casein Hydrolysis (Milk Agar); clear zone around growth, no zone round growth
Hydrolysis Test, tests for amylases. Positive, _____________. Negative, ___________
Starch Hydrolysis; clear zone around growth, no zone round growth, media brownish
UV light divided into 3 groups:
1) UV-A, longest wave, 315 to 400
2) UV-B, wave between 280 to 315
3) UV-C, wave from 100 to 280 (most detrimental to bacteria)
Which one is most detrimental to bacteria? UV causes death by causing DNA damage in the form of _______________
UV-C; pyrimidine dimers
This tests effectiveness of antibiotics on bacteria. It utilizes this agar to meets its standard requirements. If inoculated bacterium is susceptible to antibiotic, it will produce _________________. Size of it depends on bacteria sensitivity to ________
Kirby-Bauer Method; Mueller Hinton Agar; zone of inhibition; Minimum Inhibitory Concentration
Kirby-Bauer Method tests against following bacteria:
1) E.Coli
2) Proteus vulgaris
3) Pseudomonas aeruginosa
4) Staphylococcus aureus
During Kirby-Bauer Method, ________ and __________ will be white or cream-colored lawn; _________ will be yellow; __________ will be green
E.Coli and P.Vulgaris; P.aeruginosa; S.aureus
3 major Staphylococcus pathogens:
S.auerus, S.epidermis, S.saprophyticus
- most pathogenic of Staphylococcus
- can cause Toxic Shock Syndrome
- definitive test is ___________
- color is yellow, positive in everything
- This tests for its identification
- has 2 proteins: __________ and __________
S.aureus; Coagulase Plasma; Bacterial Agglutination; coagulase, protein A
- color is white
- negative in everything
- most likely won't ferment mannitol
- organism whose color is white
- negative in everything except mannitol fermentation
-selective media
- contains mannitol, 7.5% NaCl
- favors colony pigmentation
Staphylococcus Medium (SM110)
3 major types of hemolysis
1) alpha - partial lysis of red blood cells to discolorization to green
2) beta - complete lysis results in clear zone
3) y - no lysis
- cocci shaped
- forms chains of organisms when growing
- can cause meningitis, pneunomia
3 medically significant species of Streptococci
1) S.pyrogenes - strep throat, Scarlet fever
2) S.agalactiae
3) S.pneumoniae - pneumonia, memningitis
7 major groups of Streptococci
A-D enterococci, D non-enterococci, viridians, pneumococci
3 tests for alpha-hemolytic Strep.
1) Bile Esculin Agar slant (6ml)
2) 6.5% NaCl Tolerance Broth (5ml)
3) Optochin Sensitivity
3 tests for beta-hemolytic Strep.
1) CAMP Reaction
2) Bacitracin Susceptability
3) SXT Susceptability
This agar differentiates Strep species. It contains _______, which provides nutrients, _______, which inhibits growth of Gram-positive organisms, and _________, an indicator. It indicates Strep belongs to Group ____: has 2 subgroups, ________ (S.faecalis
Bile Esculin Agar Slant (6ml); Bile; Esculin; Ferric citrate; D; D enterococci, D-nonenterococci
tests ability of some Strep. to grow in 6.5% NaCl. Its pH indicator is the color ____________, and if this color changes to yellow a ________ result occurs; it differentiates Group ___ from Group ____
6.5% NaCl Tolerance Broth; purple; positive; D; non-D
This test differentiates pneumococci from viridians; positive test indicated by zone of inhibition
Optochin Susceptability
This test is positive by indication of formation of arrowhead; indicates Strep belongs to group ___
CAMP Reaction; B
This test has 2 limitations, positive result by any zone of inhibition; mostly indicates Strep belongs to group ___, but 5% group ___ and 10% group ___
Bacitracin Susceptability; A, B, C
Group A and group B are resistant to this test. It distinguishes these groups from non-A and non-B groups
SXT Susceptability
anaerobic process in which sugar is converted to alcohol by CO2 and yeast (_______________)
Fermentation; Saccharomyces cervisiae
C6H12O6 --through yeast-- 2C2H5OH + 2CO2
Reaction of Fermentation
4 types of flagellar arrangements:
1) amphitrichous (at both ends)
2) Lephotrichous (3 at one end)
3) Monotrichous (single at one end)
4) Peritrichous (all around perimeter)
3 types of movement in bacteria
1) gliding
2) rotary
3) flagellar
this genus produce endospores
Bacillus (aerobes) and Cloistridium (anarobes)
in spore staining, ___________ is primary stain, ____________ is counterstain
Malachite green, Safranin

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