LAB MIDTERM Flashcards
Aseptic technique
using practices and procedures to prevent contamination from pathogens + maintain pure cultures
Culture Media
a mixture of substances that promotes and supports the growth and differentiation of microorganisms
- general purpose (supportive)
- enriched
- minimal media
- selective
- differential
FUNCTIONAL Types of Culture Media (5):
General-Purpose Media (supportive)
Functional Type of Culture Media: support the growth of microorganisms
Enriched Media
Functional Type of Culture Media: general purpose media supplemented with highly nutritious substances such as blood
Minimal Media
Functional Type of Culture Media: contains the minimal necessities for growth of the wild-type; only contains inorganic salts, a simple carbon source, and water
Selective Media
Functional Type of Culture Media: favor the growth of some microorganisms and inhibit the growth of others
Differential Media
Functional Type of Culture Media: distinguish between different groups of microorganisms based on their biological characteristics (ex: MacConkey agar)
only use a loopful of the liquid media on the slide
To make a bacterial smear from a liquid media, you should …
first put a loopful of water on the slide and then mix in the solid media
To make a bacterial smear from a solid media, you should…
- kills the bacteria in the smear
- firmly adheres the smear to the slide - allows the sample to more readily take up stains.
Heat-fixing importance:
removes any excess water; if extra water is not removed and the smear is heat fixed, the water can boil and cause the bacterial cell to rupture and cause altered cellular morphology and arrangement – can lead to improper staining and visualization
Air-drying Importance:
Simple Stain
when a bacteria is stained with only one stain; only 2 steps (1. Staining step + 2. Washing step); fast to perform; allow microbiologist to view the shape and morphology (arrangement)
Differential Stain
staining that uses more than one chemical stain to display the differences in physical and chemical properties of different groups/types of bacteria
- shape
- margin
- elevation
- size
- texture
- color
- opacity
characteristics of Bacterial Morpholoy (7):
- circular
- irregular
- punctiform
- rhizoid
Shapes of Bacterial Morphology (4):
circular
shape: any round colony regardless of type of margin
irregular
shape: not circular; may be spreading
punctiform
shape: forming pinpoint colonies
Rhizoid
shape: root-like; elongated and branching
i. Entire (smooth)
ii. Undulate (wavy)
iii. Lobate
iv. Filamentous
v. Curled: concentric
vi. Scalloped
Margins of Bacterial Morphology (6):
i. Flat
ii. Raised
iii. Convex
iv. Raised
v. Umbonate
vi. Crateriform
Elevations of Bacterial Morphology (6):
i. Dry
ii. Moist
iii. Viscid (stick to loop)
iv. Mucoid (mucus-like)
Textures of Bacterial Morphology (4):
i. Opaque: NOT clear
ii. Translucent: clear
iii. Iridescent: shine
Opacities of Bacterial Morphology (3):
Gram-positive
purple cocci; have a THICK peptidoglycan layer that traps the crystal violet-iodine complexes and causes them to retain the purple pigment of the stain
Gram-negative
pink bacilli; have a thin peptidoglycan layer with two cellular membranes; crystal violet iodine complexes escape the thin layer of peptidoglycan when decolorizing agent is applied and washes away; retains (pink) safranin stain
Crystal Violet
Gram Stain: PRIMARY stain =
Iodine
Gram Stain: MORDANT =
alcohol
Gram Stain: Decolorizer =
Safranin
Gram Stain: COUNTERSTAIN =
Pure Culture
a laboratory culture containing a single species of organism
CFU
“colony-forming unit;” used to estimate the number of viable cells
colony forming unit
CFU =
i. Filiform: straight
ii. Arborescent: tree-like
iii. Beaded
iv. Echinulate
v. Diffuse: spreading
vi. Rhizoid
Growths on Agar Slants (6):
i. Clear: no growth
ii. Turbid: cloudy
iii. Flocculent: spread equally throughout broth
iv. Pellicle: growth at top of broth
v. Sediment: growth at bottom of broth
Growths in Nurtient Broth (5):
determines whether or not bacteria produce gelatinase and hydrolyze gelatin
Purpose of Gelatin stab =
liquid media
Positive reaction of gelatinase production:
1) Inoculate nutrient gelatin with bacteria
2)Incubate culture for 48 hours at 35 °C.
3) Refrigerate for 20 minutes.
– If media is liqiuid = bacteria produces gelatinase –
If media is solid = bacteria does NOT produce gelatinase
How to conduct gelatin stab (2 steps):
Endospore
a dormant, tough, and non-reproductive structure produced by some bacteria in the phylum Bacillota.
allows the bacterium to produce a dormant and highly resistant cell to preserve the cell’s genetic material in times of extreme stress
Function of endospores =
Bacillus + Clostridum
Genera of bacteria that produce endospores (2):
endorspores
Schaeffer-Fulton staining test stains for _______
Malachite
Schaeffer-Fulton endospore: primary stain =
heat
Schaeffer-Fulton endospore: mordant
safranin
Schaeffer-Fulton endospore: counterstain
because it can denature/break down the protein coat, as well as the cell wall of the spore to allow stain to infiltrate the interior of the spore.
why is heat used as the mordant for the Schaeffer-Fulton endospore staining procedure?
green
pink
Endospores stain ______ and vegetative cells stain ______.
Mycobacterium
which genera of bacteria are acid-fast?
Have waxy mycolic acids in their walls
what causes cells to be acid-fast?
Carbolfuchsin
Acid-fast Stain: primary stain =
acid-alcholol
Acid-fast stain: decolorizer
methylene blue
Acid-fast Stain: counterstain =
pink
blue
Acid fast bacteria appear ______ and non-acid fast facteria appear ______.
Capsule
a polysaccharide layer that lies outside the cell envelope
protect a bacterial cell from ingestion and destruction by white blood cells (phagocytosis)
function of a capsule =
negative staining (staining around the cells)
Capsules do not absorb most basic dyes so _________ _________ is used.
halos
Capsules appear like clear _____ around the cell after negative staining.
Maneval’s A and B
what reagents do we use to observe CAPSULES?
Maneval’s A
capsule visualization: acetic acid; lowers the pH in the sample and causes the Congo red to change from a red color to blue
Maneval’s B
capsule visualization: acid fuschin; interacts with the bacterial cell, staining the cell bright red
- Hanging Drop Method
- Soft-Agar Stabbing Method
methods used to see motility (2):
Motile bacterium were moving in ALL directions (Brownian movement which is NOT motility occurs when non-motile cells were vibrating in one direction)
how did you observe motility in the Hanging Drop Method?
Motile bacterium swam along the stab line AND away from it (causing the growth to appear cloudy and diffuse away from the stab line).
how did you observe motility in the Soft-Agar Stabbing Method?
MOTILE
is E. coli motile or non-motile?
NON-motile
is M. luteus motile or non-motile?
selective + differential
functional type of media of MAC (MacConkey Agar)
selective
functional type(s) of media of PEA (Phenylethyl Alcohol Agar):
enriched + differential
functional type(s) of media of blood agar
negative
Gram-________ can grow on MAC
positive
Gram-________ can grow on PEA
both
can Gram-negative or positive grow on blood agar?
lactose fermenters vs. non-lactose fermenters
what does MAC differentiate between?
RED
no color change
MAC:
appearance of lactose fermenters:
appearance of non-lactose fermenters:
hemolytic (can digest/hydrolyze blood) vs. non-hemolytic
what does blood agar differentiate between?
clear zone
appearance of BETA-hemolyic bacteria in blood agar =
green
appearance of ALPHA-hemolyic bacteria in blood agar =
NO clearing
appearance of GAMMA-hemolyic bacteria in blood agar =
Conducted the bacterial growth curve by measuring + plotting the bacterias’ absorbance every 20 minutes using a spectrophotometer
how did we conduct and graph the bacterial growth curve?
- Lag Phase
- Exponential Phase (Log)
- Stationary Phase
- Death Phase
Phases of Bacterial Growth (4):
lag phase
phase of bacterial growth: occurs when cells are placed in a fresh medium; population size remains temporarily unchanged
exponential (log) phase
phase of bacterial growth: cells are regularly dividing by binary fission; rate of growth is expressed as “generation time” and “doubling time”
stationary phase
phase of bacterial growth: population of cells stops increases and remains constant
death phase
phase of bacterial growth: viable cell population declines
a. Obligate Aerobe
bacteria that NEED oxygen (aerobic environment) to survive; found at the TOP of the FTM tube
Obligate Anaerobe
bacteria that live in an anaerobic environment and cannot survive in the presence of a lot of oxygen; found at the BOTTOM of the FTM tube
Facultative Anaerobe
bacteria that are ABLE to survive in an anaerobic environment, but PREFER to live in an aerobic environment with oxygen; found mostly at the top of the FTM tube with “tapering” growth
Microaerophile
bacteria that survive within a NARROW range of oxygen concentration; looks like a STRIP of growth slightly below the top of the FTM tube
Aerotolerant anaerobe
bacteria that live in an anaerobic environment (without oxygen) but can TOLERATE the presence of oxygen; found at ALL areas of the FTM tube
Acidophiles
microorganisms that grow at a pH near 3
Neutrophiles
microorganisms that grow at a pH near 7
Alkaliphiles
microorganisms that grow optimally at a pH between 7.5 and 11.5
Water Activity
amount of unbound water in a sample; based on a scale of 0 to 1
1
water activity of pure water =
higher
The _______ the water activity, the faster that microorganisms like bacteria, yeast and mold will be able to grow.
hypotonic environment
water comes INTO the cell
hypertonic environment
water LEAVES the cell
Halobacterium salinarium
of the bacteria that were tested, which grows at the HIGHEST salt concentration?
to isolate a single colony from a bacterial culture
purpose of streak plate
preparing areas of dilution – streak an area once, then go over one line of the streak over and over again until it is diluted enough for one colony to grow by itself
how do you perform a streak plate?
Primary Containment
concerns the protection of personnel and the laboratory environment from exposure to infectious microbes
Secondary Containment
deals with protecting outside environment from exposure to infectious organisms; depends on design of laboratory and availability of equipment; workers should maintain labs safety features
