Mid Term Practical Flashcards
A species of pathogenic aerobic endospore forming bacterium is ____ and causes _______
Bacillus anthracis + anthrax
A species of anaerobic endospore forming bacterium is ____ + causes _____
Clostridium tetani + tetanus
Capsules may be made of
polysaccharides, glycoprotein, or polypeptides
Capsules may be made of
polysaccharides, glycoprotein, or polypeptides
Negative stains stain
everything except what you want to see
At the end of the endospore staining process
Endospores are green and vegetative cells are red/pink
At the end of the endospore staining process
Endospores are green and vegetative cells are red/pink
In acid fast stains
non-acid fast stains blue
acid-fast stains pink
What is used in acid-fast stain?
Carbol fuschin (2 min)
Acid alcohol
Methylene blue (1 min)
What is used in capsule stain?
Crystal violet (2 min)
Copper sulfate
What is used in endospore stain?
Malachite green (warmer) (5 min)
Malachite green (cooling) (5 min)
Safronin (2 min)
Who developed acid-fast stain?
Paul Ehrlich
Name of acid-fast used in the lab
Kinyoun
2 pathogenic species of mycobacterium are
Mycobacteria tuberculae + leprae
TB + leprosy
What parasite can be identified by acid fast?
Cryptosporidium and toxoplasma
Mycobacterium is filamentous (like a treebranch) and Nocardia is rod shaped
False
Where do used glass test tubes discard?
Discard rack
Where do used glass slides go?
Used slide basin
Where does broken glass go?
Broken glass container
Where do used latex gloves go?
Autoclave trash
Where do paper towels used in bench disinfection go?
Regular trash
Where does uncontaminated trash go?
Regular trash
Where do contaminated Petri plates go?
Autoclave trash
Where do qtip swab go?
Regular trash
How to calculate total magnification of an object?
Ocular (10x) x objective
Purpose of immersion oil
Decreases light refraction and loss of light
Define ubiquitous
Found everywhere
Define pure culture
Single genus + species
Define colony
Single bacteria divides by binary fission and grows large enough to be seen by naked eye
Define turbidity
Cloudiness which indicates growth
Define media
Nutrient material suitable for growth of microorganisms
Define innoculum
Sample of microbial culture
Define aseptic technique
Safely moving microbes without contaminating environment, oneself, or the microorganism
How much is in a pinpoint amt of bacteria?
1 mil
Why are agar plate labeled on bottom and inverted when incubated?
So condensation doesn’t disrupt colony formation
State the criteria used to describe a bacterial colony
Shape
Margin
Elevation
Colony
Order of gram stain
- CV (1 min) DIH2O
- Iodine (mordant) (1 min) DIH2O
- Acetone-alcohol (2-5 sec)
- Safronin (1 min) DIH2O
What color should Gram + and Gram -?
Gram + : purple (cocci)
Gram - : pink (bacilli)
What stains can show size, shape, and arrangment?
Simple and Differential
Two genera that can be seen in an acid-fast stain?
Mycobacterium + nocardia
What color should acid-fast and non-acid-fast bacteria?
Acid fast: Pink
Non acid fast: Blue
Two diseases caused by acid-fast bacteria?
TB + leprosy + nocardiosis + cyptosporidiosis
Why are acid fast bacteria acid fast and why do they stay pink?
The bacteria have a waxy wall made of mycolic acid that traps the carbol fuschin in. It resists decolorizing by acid-alcohol.
Steps in acid-fast stain?
- Carbol fuschin on filter paper (2 min on heat) DIH2O
- Acid alcohol (2-5 sec off heat) DIH2O
- Methylene blue (2 min off heat) DIH2O
What are 2 genera of bacteria that produce endospores?
Bacillus and clostridium
Endospore stain process?
- Malachite green w/filter paper (5 min on warmer)
- Malchite green w/ filter paper (5 min off warmer) DIH2O
- Add safronin (2 min) DIH2O
Difference between neg. stain and pos. stain:
Positive (+) Stain: stains everything you want to see
Negative(-) Stain: stains everything except for what you’re trying to see
Why won’t capsules hold a stain?
Capsules have a neutral charge and stain have a positive charge
Why don’t we heat fix negative or capsule stains?
Heat will denature the protein in the capsule and you won’t be able to see it
Bacteria that grows in the candle jar is:
Microaerophile
Capnophile
The two major types of microscopes that have been developed are the
Compound and electron microscope
What are the 4 types of microscopes?
Phase contrast
Brightfield (we use these)
Darkfield
Fluorescent
Two factors in determining how well you’ll see through the microscope is:
The magnification and
Resolving power
Define resolving power
Resolving power is measured by how well your microscope can show two lines lying close together as two distinct lines
Define refractive index
How well the lens is able to bend light
State the function of the microscope condenser
concentrates the beam of light on the specimen
State the function of the microscope iris diaphragm
Regulates the intensity of light entering the lens
State three steps to achieve adequate lighting when the oil immersion objective is used to view a specimen
The condenser is raised to the highest position.
The iris and base diaphragm are open.
The rheostat is used to regulate light intensity
The two components that influence the resolving power of a microscope are the
Wavelength of light
Numerical aperture
As you go from low to high-dry objectives, the diameter of the iris diaphragm
Decreases
The refractive index of glass and immersion oil are the same
True
TSA(trypticase soy agar) is an example of nutrient media to grow bacteria
True
What are the damaging by-products of oxygen during aerobic energy production (cellular respiration)?
Superoxide and peroxides
Name three enzymes that neutralize the damaging by-products of oxygen during aerobic energy production (cellular respiration).
catalase, peroxidase, superoxide dismutase
How does the Gas Pak jar achieve an anaerobic atmosphere?
The added packet of chemicals generates hydrogen. The hydrogen binds oxygen to produce water.
How does the Candle Jar achieve an atmosphere to grow microaerophilic, capnophilic organisms?
The flame consumes oxygen and releases carbon dioxide
chemicals produced by some bacteria and fungi that, in small quantities, can inhibit the growth of bacteria
Antibiotics
the lowest concentration of an antibiotic that can inhibit the growth of a test organism
Minimal Inhibitory Concentration (MIC)
E-test
Combination of Kirby-Bauer and Minimal Inhibitory Concentration tests
Disadvantages of “broad-spectrum” antibiotics?
Broad spectrum antibiotics are probably contributing to the escalating drug resistance we are seeing in microorganisms. Broad spectrum antibiotics often wipe out a person’s normal microbiome as well as the pathogen they are intended to kill, resulting in superinfections from organisms
What are some organisms that can cause super infections?
Clostridium difficile and Candida albicans
List the factors that must be controlled to standardize the Kirby-Bauer test pt. 1
-stability of the antibiotic
-the rate of diffusion of the antibiotic
-the bacteria being tested
-the pH of the culture medium
-the depth of the culture medium
List the factors that must be controlled to standardize the Kirby-Bauer test p. 2
-the inoculum density
-the incubation time
-the incubation temperature
-the concentration of the antibiotic
What kind of agar plate do we use for Kirby-Bauer testing?
Mueller-Hinton Agar Plate
E. coli and Pseudomonas are gram ____ bacteria?
Gram negative
Staphylococcus epidermidis is gram _____ bacteria?
Positive
Negative Beta-Lactamase Result
-No pH change=organism did not produce beta-lactamase.
-Organism is sensitive to penicillin
-Turns PINK
Positive Beta-Lactamase Result
-pH change
-Organism produce beta-lactamase
-Organism is resistant to penicillin
Which gene allows jellyfish to glow in the dark?
GFP
able to take up the DNA (e.g. plasmid) from the environment
Competent Cells
Define “competent” cells
Able to pick up DNA from the environment
What laboratory procedure is done to encourage cells to become competent?
growth in the presence of calcium chloride
What is the relationship between a gene and a protein?
A gene is a piece of DNA which provides the instructions for making a protein
Compare “chromosomal DNA” and “plasmid”
Chromosomal DNA contains essential information for bacterial growth and reproduction. Plasmid DNA are small, circular DNA separate from its chromosome which contains non-essential infornation
List the genes contained in the pGLO plasmid
GFP, BLA, Ara-C
How is the expression of the Green Fluorescent Protein gene regulated?
The BLA gene and Ara-C gene are continually transcribed and translated into protein. The gene for GFP, on the other hand, is regulated by the Repressor protein and will not be transcribed without the addition the sugar arabinose to the cells’ nutrient medium. The arabinose will inactivate the repressor protein thus allowing the GFP gene to be transcribed.
Genes can be transferred from one organism to another by
Transformation
Aa plasmid that contains the gene for green fluorescent protein and resists ampicillin
pGLO
Able to take up a plasmid
Competent bacteria
Explain why certain organisms are “acid-fast”
Waxy wall enables the bacteria to resist decolorizing with acid alcohol
What is the difference between the Ziehl-Neelsen stain and the Kinyoun stain?
Ziehl-Neelsen stain uses steam Kinyoun uses a concentrated dye, heat, and longer stain time
List four acid fast organisms and their associated disease names
Mycobacterium tuberculosis-tuberculosis
Mycobacterium leprae-leprosy or Hansen’s disease
Nocardia asteroides-nocardiosis
Cryptosporidium-cryptosporidiosis
Name the dyes used in the Schaeffer-Fulton endospore stain
Malachite green and Safranin
Define vegetative cell
Metabolically active, replicating cell
Define vegetative cell
Metabolically active, replicating cell
Media used for antibiotic sensitivity test
Mueller-Hinton
Media used for antibiotic sensitivity test
Mueller-Hinton
Genes located on the pGLO plasmid
BLA, GFP, ARA-C
Ara-C protein is a
Repressor protein that regulates transcription of GFP gene
Beta-lactamase does what?
Breaks apart ampicillin (targets beta-lactam ring)
GFP is
Gene that translate to become fluorescent protein that glows under UV light
Ara-c block what?
Transcription of GFP
What does arabinose do to Ara-c?
Inactivates it and allows RNA polymerase to transcribe and translate the GFP protein
Difference between slime layer and capsule
Slime layer: loosely bound
Capsule: tightly bound, easier to visualize
Capsule in virulence
Decreases phsagocytosis, aids in attachment, can be a source of nutrition, helps prevent drying
State two differences between the smear preparation for a Gram stain and a capsule stain
Do not heat fix
Do not rinse with water
Anthonys capsule stain method
Stains the background blue
Most common stains in capsule/negative stain
Nigrosin and india ink
How does the thioglycollate broth grow anaerobic bacteria as well as aerobic bacteria?
Agar prevents diffusion of oxygen into the medium. Thioglycollate binds oxygen. Together they produce a decreasing level of oxygen in the media.
Methylene blue is blue in color in the presence of oxygen
True
