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Biochem Practical
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97
Biology
Undergraduate 3
10/12/2009

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Term
Brightfield microscopy
Definition
Blue (daylight filter)

observation of unstained specimens
Term
phase contrast microscopy
Definition
Green filter
Push slider far L or R at 100x
Push slide from opp unmarked end for 40 x
Term
Heat Fixing
Definition
pass smear slide thru hottest part of flame (blue), twice- cells then adhere to the slide
Term
Microbial Eucarya include:
Definition
algae
protozoa
fungi
slime molds
Term
Common shapes of Bacteria and Archaea
Definition
spherical
bacilli (rod shape)
spirilla (Spiral)
Term
Protozoa shape
Definition
wide variety
Unicellular
Term
Fungi
Definition
Filamentous(molds)
unicellular (yeast)
Term
Algae
Definition
unicellular or multicellular
wide variety of shapes
Term
Types of Bacteria
Definition
Psudomonas sp.
Bacillus sp.
Staphylococcus sp.
Enterococcus sp.
Term
Types of Microbial Eukarya
Definition
Saccharomyces sp.
Penicillium sp.
Term
Ocular micrometere Calibration
Definition
use stage micrometer .01 mm- 10 um
Term
Calibration Factor
Definition
number of stage micrometer divisions that corresponds to one ocular micrometer division

Measured in um/ocular unit
Term
Direct Cell Count (Hemacytometer):
1. Advantage
2, Disadvantage
Definition
1. the cells are actually observed and then counted
2. difficult to distinguish between viable and non-viable cells
Term
When cells fall on a line during direct count you should:
Definition
Include those on the top and left hand lines
Exclue those on the bottom and right-hand lines
Term
Connected chains and clusters in direct count technique is counted as:
Definition
1 one cell unit
Term
Units of Av. # cells per small square
Definition
cells per mL
Term
most bacterial dues are salts of two types:
Definition
acidic or basic
Term
Acidic Dyes
Definition
bear colored portion of the anion
was combined with a base, NaOh, form water solube salt.
Term
Basic Dyes
Definition
bear chromophore on the cation
formed by combin. of color bearing compound w/ an acid like HCl form water soluble salt
Term
Types of Basic Dyes
Definition
Methylene blue
basic fuchsin
crystal violet
safranin
Term
How do bacteria react toward stains?
Definition
Cells act as if they bear a net negative charge, therefore a basic dye is used and binds firmly to the negative cell
Term
Simple Stain
Definition
flood dried heat fixed smear w/ stain and allow to sit for ~ 1 min and rinse gently with water, observe.
Term
Negative Stains
Definition
Colors the background but leaves the cells unstained and transparent, so they appear as light areas on a darkened field
Term
What kind of dyes are used for Negative Stains
Definition
Acidic dyes like nigrosin or neutral dyes like india Pink since they do not readily stain the cells

Used commonly for Spirochetes
Term
Differential Stain
Definition
Stains that involve differences in cell wall composition:
Gram Stain and Acid-Fast Stain
Term
Gram Stain
Definition
Requires use of Four Reagents:
1.Crystal Violet (B.D.)- Stains the Org.
2.Iodine (Mordant)- Increases affinity between cell and the dye
3.Ethanol (D.A)- washes dye out of cell
4.Safranin (Counterstain) (B.D.)- gives the decolorized cells a contrasting color
Term
Gram Positive
Definition
Cells retain initial dye (Crystal Violet) and appear purple
Term
Gram Negative
Definition
Cells that decolorize readily and appear red after counterstain (pink)
Term
Gram-Variabe
Definition
cells that are intermediate between the gram + and gram - stains.
Term
Acid-Fast Stain
Definition
differential stain, used to distinguish between organisms that contain very large amounts of waxy lipid material (mycolic acids)
Term
Endospore Stain
Definition
Structural staining that is used to distinguish the presence of the bacterial endospore. Flood the smear with Malachite green dye, and then heat slide. New technology allows use of red carbol fuchsin and methylene blue
Term
Capsule Stain
Definition
reveals slimly layer that surrounds bacteria, capsule stains less readily then the rest of the cell. Combination of simple and negative stain used so the background is dark with the - stain, the capsule is clear and bright around the colored cells.
Term
Who discovered Gram Staining and when?
Definition
Christian Gram in 1883
Term
Gram + and Gram - Staining indicates:
Definition
Cell wall type only for Bacteria
Term
What is the Critical step in Gram Staining
Definition
Step Three:
Use 95% Ethanol, tilt slide gently and add drop by drop slowly for 20 s or until most of the dye has washed off the smear
Term
Chemically defined (Synthetic) Media
Definition
Prepared w/ precise chemical composition known. Used for enrichments [exercise 7] or in vitamin assay work.
Term
Complex (non-synthetic) media
Definition
Prepared where exact composition is NOT known, such as meat extracts, enzymatic digests of proteins, and yeast extracts. Support growth of wide variety of microorganisms.
Term
General Purpose Media
Definition
Support growth of Chemoheterotrophs
Ex- Nutrient agar has beef extract, peptone and agar in water.
Term
Fastidious Chemoheterotrophs
Definition
must be cultured on general purpose media that has been enriched or supplemented with particular growth factors.
Term
Differential Media
Definition
Contains reagents or chemicals that produce differences in growth and allow an observer to differentiate between types of microbes.

Ex- Blood agar medium, some bacterial colonies hemolyze the blood cells and produce clear areas in the agar.
Term
Selective Media
Definition
Allow growth of only certain types of organisms due to addition of inhibitors or a specific carbon source.

"Survival of Fittest"
Term
Ingredients commonly used in media
Definition
sodium chloride
hydrochloric acid
beef extract (amino acids, vitamins, minerals)
Peptone
Yeast Extract (AA, NA, GF)
Term
Agar
Definition
complex carbohydrtae material of marine algae used ot produce semi-solid media!
Solidfies at 40-45 C and will not remelt until boiled.
Only some MARINE microbes use it as a nutrient material
Term
Who invented the Enrichment Culture Technique
Definition
19th Century Beijernick (Holland) and Winogradsky (Paris)
Term
What is the Enrichment Culture Technique
Definition
allows isolation of almost any kind of organism using a liquid medium and providing suitable growth conditions
Term
Beijerinck Experiment
Definition
Used liquid medium that contained nutients, good carbon source, energy source, most minerals, lacked nitrogen compounds. Used canal water, favored growth of organisms that utilized atmospheric nitrogen - NITROGEN FIXATION.
Term
Common type Enrichment provides
Definition
essential nutrients with one specific carbon and energy source (C/E) such as cellulose or petroleum hydrocarbons.
Term
Experiment 7- Enrichment Culture Technique uses what type of media?
Definition
General Purpose growth medium
Term
What macronutrients are used in Ex 7 E.C.T. and the pH?
Definition
MgSO4
CaCl2
KH2PO4
K2HPO4
NH4NO3
FeCl3

pH of 7
Term
What was added to the media to reduce the growth of fungi and enhance the ability of recovering bacteria in Ex 7 ECT?
Definition
Cycloheximide (an antibiotic inhibits 80S ribosome function)
Nystatin (Antibiotic that inhibits function of fungal plasma membranes.)
Term
Single Viable Cell deposited on Solid Agar Medium produces:
Definition
Reproduces to form a cluster or colony of cells
Term
Isolated Colony
Definition
Constitutes a pure cultures, because it is considered to consist of similar cells all derived from the same original cell.
Term
To make a detailed study of an organism this is needed:
Definition
Isolated colonies can be obtained to produce PURE CULTURE
Term
List two types of Selective Media used in Ex 8 of pure culture technique and selective media
Definition
Mannitol Salt Agar (MSA) - 10% Sodium Chloride as its key selective factor.
MacConkey Agar (MA)- Contains bile salts and crystal violet dyes as the key selective factor.
Term
Mannitol Salt Agar (MSA)
Definition
bacteria can grow and ferment Mannitol
Form Yellow colonies
Those that can grow but CANNOT ferment mannitol form Red/Pink Colonies,
Term
MacConkey Agar (MA)
Definition
Bacter that can grow and ferment lactose form pink-dark red colonies
Those that can grow but CANNOT ferment Lactose form colorless colonies.
Term
Pour Plate technique
Definition
small sub sample is inoculated into sterile petri plate and mixed with melted general purpose agar, it hardens and is incubated at app. Temperatures. During this each viable cell grows into a mass of cells and these masses appear as visible colonies
Term
Spread Plate Technique
Definition
used to prepare a plate in which all colonies appear on the surface (used w/ Aerobes) Created by inoculating the sub-sample onto the surface of a pre-poured, cooled plate
Term
To ensure cell replication does not occur dilution blanks should be placed
Definition
in chilled water baths before mixing the dilutions and agar together
Term
How do you mix the contents of a pour plate?
Definition
By rotating plate in a gentle figure-eight motion on the lab bench before the agar cools
Term
What is added to a spread plate to assist in distribute the sample evenly?
Definition
several 4-5 glass beads
Term
In the case of a sample containing spreading organisms this can be added
Definition
sterile agar, small amount poured over the plate after it cools.
Term
Instruments used for counting in Ex. 9 Enumerations of microorganisms
Definition
pen/ light box
Quebec Colony Counter
Term
Only plates yielding___ to____ colonies are selected for counting
Definition
30-300
Term
Standard plate count is reported as
Definition
the number of viable organisms or" Colony-Forming Units" per gm, or per mL of sample
Term
Report plates with no colonies as
Definition
less than one
(<1)
Term
Report plates with fewer than 30 colonies as
Definition
Count teh actual number of colonies on teh lowest diluted plated, divide by appropriate dilution used and report as the pate count.
Term
Spreading colonies
Definition
1. not uncommon, if more then half the surface of the plates is covered but no CFU/plate values, report as spreaders (Spr)
2. if covers over half report as greater then 300 colonies/plate.
3. laboratory accident- contamination, missed dilution
Term
R2A Contained
Definition
yeast extract, peptone, glucose, casamino acids, soluble starch
Term
Target Microbes for R2A medium
Definition
Chemoheterotrophic Bacteria
Term
Violet Red Bile
(VRBA)
Definition
Yeast extract, peptone, lactose, bile salts, neutral red, crystal violet.
Term
Target Microbes for VRBA medium
Definition
Gram - enteric bacteria able to degrade lactose and withstand the presence of bile salts and dyes
Term
What technique was used with R2A Agar
Definition
pour plate and spread plate
Cycloheximide was added to inhibit fungal growth
Term
Spread plate technique was used in Ex 10 to distinguish
Definition
morphotypes- descriptions for form, elevation, and margin, colony pigmentation and surface texture.
Term
Morphotypes
Definition
1. colonies with diff morphotypes are NOT the same type of bacteria
2. Colonies with the same morphotype may or may NOT be the same type of microbe.
Term
Diversity Indices
Definition
Used to indicate community structure and stability
Term
Simple Diversity calculation
Definition
Species Richness- d

d- (S-1)/log N

S= # of species (colony types)
N- # of individuals (total # of colonies at a spec. dilution)
Term
Common Diversity Index
Definition
Shannon-Weaver Index of Diversity (H)

H=(C/N) ((N LOG N -(SUM ni log ni))

C-2.3
N= # colonies
ni=# colonies/colony type
Term
Highly selective Medium used in Ex 10 microbial enumeration
Definition
VRBA
due to the inhibitory bile salts and crystal violet dye, which will keep most non-enteric gram negative bacteria as well as gram positive bacteria from growing.
Term
VRBA was used with food and water samples as a check on possible_______ ______
Definition
fecal contamination
Term
What technique is used for VRBA
Definition
Pour plate due to fact that all enterics can grow in absence of oxygen and all coliforms can grow in presence of oxygen
Term
R2A Agar samples incubated at
Definition
30 C
Term
VRBA agar was incubated at
Definition
35-37 C
Term
In R2A agar you counted ___ colonies
Definition
ALL
Term
In VRBA agar you counted only _____ colonies
Definition
coliform (red/purple), Gram Negative enteric bacteria able to produce acid from lactose fermentation
Term
Morphotype: Forms
Definition
punctiform, circular, filamentous, irregular, rhizoid, spindle
Term
Morphotype: Elevations:
Definition
Flat, raised, convex, pulvinate, umbonate
Term
Morphotype: Margin
Definition
Entire, Undulate, Lobate, Erose, Filamentous, Curled
Term
Calibration factor for 10x
Definition
10 um (Micrometers)
Term
Calibration factor for 40 x
Definition
2.5 um (micrometers)
Term
calibration factor for 100x
Definition
1 um (micrometer)
Term
E Coli is
Definition
Gram - and looks like short fat rods
Term
Staphylococcus aureus is
Definition
Gram + and clonies were small white and cocci
Term
Colonies that appear yellow on MSA
Definition
can ferment mannitol
gram + Cocci
Term
Colonies that appear red/pink on MSA
Definition
cannot ferment mannitol
Gram + Cocci
Term
Colonies that appear pink- dark red on MA
Definition
can ferment lactose
Gram - Rods
Term
Colonies that appear colorless on MA
Definition
Cannot ferment lactose
gram - rods
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