OSU Microbiology 4000: Lab Practical. Everything.
Autotrophic
produce own food source from inorganic carbon and energy sources.
Bacteriostatic
Antibiotic that inhibits bacterial growth.
Precipitin Test steps
(BSA) Antibody in 1 capillary tube, then draw up antigen from another capillary tube then place in clay and see what precipitates.
Trypticase Soy Agar
(TSA Plate) general plate with many nutrients. innoculate at 30 deg C. Same temp as body, helps with growth
prokaryotes
(do not have visible cilia and flagella under a light microscope) Cells that do not contain nuclei
eukaryotes
(have visible cilia and flagella under a light microscope) Cells with internal membranes
Constructive milk plated
Buttermilk
alkaliphile
microbes that show optimal growth at high pH
Current flow
where the movement of all objects are in one direction due to a current
Brownian motion
where the objects bounce around in all random directions and vibrate around
petri dishes, used gloves, contaminated paper towels
white biohazard container
Can E. coli survive on MM1? Z. salivarius? S. epidermis?
yes, no, no
Objective's we used in lab
40, 10, 100
nonhalophiles can grow in
0-2%
Describe the KIA (6)
1 red slant and a yellow but is glucose fermentation, 2 lactose and glucose fermentation is yellow slant and yellow but 3 protein utilization is a red slant and no change in butt (staying orange) or red throughout. 4 Gas is produced in the yellow slant, yellow butt, cracking the agar. 5 in red slant yellow but a black precipitate can form signifying desulfhydrase activity creating FeS. 6. remaining orange is nothing utilized.
Magnification of Compound bright field microscope
10 (ocular magnification) x ? (objective magnification)
Total magnification we used in lab
100, 400, 1000
How many times do you use buffer in ELISA
3 steps involve washing with wash buffer
What was the best antiseptic?
3.0% H2O2
Size of paramecium
50-300 micrometer eukaryote, protazoa
endospore
A thick-walled protective spore that forms inside a bacterial cell and resists harsh conditions. (Bacillus and Clostridium, undergone sporulation, therefore can survive even prolonged exposure to moist heat at relatively high temperature)
Differential Acidic dyes
Acid fuchsin
Bactericidal
Antibiotic that can kill bacteria.
Agglutination reaction
Ag-Ab complexes form when antibodies react with insoluble antigens, which are located on invading cell surfaces. This reaction causes the cells to lump together, making it easier to remove the invaders from the body by phagocytosis.
Preciptin reactions
Ag-Ab complexes form when antibodies react with soluble antigens; the insoluble complexes precipitate from solution and are removed from the body either by phagocytosis or filtration.
Differential media
Allow a variety of organisms to grow, but enable the observer to visually distinguish among different types of bacteria based on how they metabolize the components of a given medium.
Thymine dimers
Alter the genetic code of a cell affecting replication and transcription, resulting in cell death.
Osmotic pressure
Amount of pressure needed to stop the net flow of water across a semipermeable membrane. Most bacterial habitats are hypotonic.
The kirby-bauer method
Antibiotics dispensed on plate, if zone of inhibition is there then the zone of inhibition is inoculated onto another plate to determine if something is bacteriocidal, or bacteriostatic.
Disinfectants
Applied topically to inanimate materials.
in the precipitin test, the soluble antigen is
BSA
Symbiotic relationship of yogurt
Bacteria make the yogurt more acidic.
microaerophiles
Bacteria that use oxygen, but only at a low concentration
Transient microbiota
Bacteria, mold spores, etc. that we have on our skin surfaces from touching objects in our environment.
What do anti rhesus antibodies agglutinate?
Blood with rhesus antigen
What do anti-a antibodies agglutinate?
Blood with the A antigen.
What do anti-b antibodies agglutinate
Blood with the B antigen
Citrate
Blue=positive, means pH change and the ability of the organism to use citrate as a carbon source.
Stomacher
Breaks up a sample using the same type of physical action that the human stomach produces.
Units for TVC?
CFU/mL
facultative anaerobes
Can make enough ATP to survive using using fermentation or respiration. (with or without oxygen)
Acid-fast bacteria
Can't be stained under normal circumstances due to mycolic acid. Technique involves primary stain, two mordants, decolorizing agent, and counterstain.
obligate anaerobes
Cannot survive in the presence of oxygen
Describe phenol red carb. broth test
Carbohydrates have been used if the solution turns yellow, it is red only nothing was used, red and cloudy then peptone was used, yellow with a little air bubble in the durham tube is gas produced and carb. used.
Plasmolysis
Cell membrane is pulled away front he cell wall, causing the bacteria to die.
IMViC
Collectively the MRVP, citrate, and indole tests
How did we test the effectiveness of ampicillin and kanamycin on transformation
Competent cells- transfer 50 microliters in plasmid A, 50 microliters into p B. - Ice for 20 minutes those two, heat shock 42 Celsius for 90 seconds, then place in ice for 2 minutes. -Plate plasmid A on half of the ampicilin plate, half of the kanamycin plate then do the same for plasmid B on the opposite side of each.
What does the UV radiation do to the bacteria to control them
Creates the thymine dimers and damages the DNA backbone.
Simple stain Basic dyes
Crystal violet, methylene blue
Differential stain Basic dyes
Crystal violet, safranin, malachite green
Bacteria that isn't stained would be viewed using
Darkfield or phase contrast.
What are examples of gram -, + and fungi in biofilm
E. coli, s. aureus, Candida Albicans.
are Catalase and Cytochrome oxidase endo or exoenzymes
Endoenzymes
Virulence factors
Enhances ability to evade host defenses and cause disease. -Capsule -Enzymes -DNase -Coagulase -Hemolysins
Nitrate reduction
Enteric facultative anaerobes in the absence of oxygen use nitrate as a terminal electron acceptor resulting in reduction of nitrate (NO3) to nitrite (NO2) or ammonia (NH3.)
ELISA
Enzyme-Linked Immunosorbent Assay. Employs the use of secondary antibodies that are conjugated to a specific detector enzyme.
Peptidases
Enzymes produced by bacteria to break down proteins into small peptides or amino acids. Some are exported from the cell into the environment where they become catalytically active.
Budding
Form of asexual reproduction used by yeast cells.
MacConkey Agar Plate
Gram-negative enterics usually grow on these plates. If the colonies are hot-pink the organism can ferment lactose and are coliforms. If the colonies are light yellowish, the organism can't ferment lactose.
Extreme thermophile
Growth at 80C or higher
Psychrotroph
Growth between 20-30C
Mesophile
Growth between 25-40C
Thermophile
Growth between 45-60C
What is hypotonic, hypertonic and isotonic
Hypertonic is a lot of solute in the environment drawing the water out. Hypo is the opposite.
Starch-agar plate
If starch has been utilized, there will be a clear area around the streak, indicating that the organism can hydrolyze starch and is alpha-amylase positive.
How are nitrogen reduction and denitrification identified in the nitrate reduction procedure?
If the tube doesn't change color after the sulfanilic acid and naphthylamine, and the zinc dust and there is a bubble in the durham tube-> denitrification. If there is no bubble -> Nitrogen reduction. Red color after the first test -> NO3- -> NO2- stop
Antibiotic modes of action (4)
Interfere with cell wall synthesis Inhibit protein synthesis Inhibit nucleic acid synthesis Negatively alter plasma membrane
Endoenzymes
Intracellular peptidases, catalytically active inside the cell.
Coarse focus knob
Larger knob on inside used to rapidly change the focus
Compound bright-field microscope
Light microscopes, specimens appear dark against bright background. "Bright field." (the most commonly used)
dark field microscope
Light source is above specimen, creating dark background and light image
What was the best disinfectant?
Lysol
Describe MR test
Methyl red is a red pH indicateor used in examining the amount of acids due to glucose fermentation. If the amount of acid is low the indicator remains yellow or stays yellow, if the pH drops the Methyl red changes the color to red.
What are antiseptics that we used
Mouthwashes
capsule stain
Negative stain - the structure of interest (capsule) remains colorless Uses acidic dye - Staining reagent Maneval's solution (acid fuchsin)
ground beef when cooked, Constructive or destructive?
Neither
Distinction between nitrogen reduction and denitrification
Nitrate reduction NO3- ->NO2- -> NH3 (ammonia) used by facultative anaerobes without the presence of oxygen. Denitrification NO3- -> bunch of steps N2-.
Isotonic
No net movement of water molecules, cell structure isn't affected by the tonicity of the environment.
Psychrophile
Optimal growth below 15C
destructive milks plated
Pasturized milk products
Exoenzymes
Peptidases exported from the cell and catalytically active outside the cell.
Describe the VP test
Pink ring signifies a positive result. This represents the use of the Butenol pathway. because the acetoin reachts with potassium hydroxide and alpha-naphthol. If it stays clear then it is a negative
gram stain (differential)
Primary stain - Crystal Violet Mordant - Gram's Iodine Destain - Acetone-Alcohol Secondary stain - Safranin
crystal violet stain
Primary stain, gets stuck in the cell wall (peptidoglycan
Destructive juice plated
Processed, frozen concentrate, fresh squeezed
Destructive processes
Produce food spoilage.
Desulfhydrase
Protein test: Another do KIA test
Gelatinase Test
Protein test: detects enzymatic breakdown of gelatin to polypeptides. and polypeptides to amino acids
cytochrome oxidase
Put the microbe on a slide and use oxidase reagent. oxidizes cytochrome C3 A dark purple color will show within 30 seconds.
Denitrification
Series of reactions to reduce nitrate to nitrogen gas (N2.) Other gaseous forms of nitrogen are also formed (NO and N2O.) Returns nitrate back to the atmosphere.
MRVP
Red ring=positive, acetone is produced and glucose is fermented using the butanediol pathway.
Tryptone
Red ring=positive, tryptophanase is produced and the organism can break down tryptophan into pryruvate, ammonia and indole.
KIA
Red=protein utilization Yellow=acid production and carbohydrate fermentation of glucose and/or lactose. Gas bubble= gas produced Black precipitate= desulfhydrase is produced
Halophiles
Require higher salt concentration, up to 20%.
Constructive processes
Result in desirable products and often involve fermentations.
2 bacteria involved in yogurt producion
S. thermophilus, lactobacillus bulgarious
Margin of a colony (6)
Smooth, Undulating, Lobed (almost villi coming out), irregular (rough), ciliate (lot's of white lines through it). filamentous (branchy)
darkfield microscopy
Species looks bright against a dark bacground.
flagellar motion
Straight lines or flagella tumbles.
Microbiology
Study of microscopic organisms that are below the limit of vision of the human eye.
What is P. polymyxa
The bacteria that produces the natural antibiotic.
Describe the Simmons Citrate test
The citrate starts out with a green medium. It changes to blue slant on top if the pH goes up and the citrate (acid) is broken down
Total viable Count
The direct quantification of the number of living cells in the culture- Enumerate with Colony forming Units/ (dilution factor*volume plated)
cellular arrangement
The patterns of cells
cell morphology
The physical appearance and behaviour of bacteria under a microscope including size, colour, gram staining and colonial appearance.
Nose piece
The rotating part of the microscope with the objectives.
Fine focus knob
The smaller knob that is used for precision focusing
zone of inhibition
The zone where bacteria can't grow around a given antibiotic. (small - resistant)
Antiseptics
Used for topical application to living tissues.
Immunofluorescent microscopy
Used to discover organisms that can fluoresce
phase contrast microscope
Uses refractory properties of light, creating a 3D image; it is good for visualizing cell organelles; "straw in water"
Normal microbiota
Usually inhabit our body and cause no harm under normal circumstances.
How does ELIZA work?
Wells are filled with wash buffer, then the first antibodies are put in, then they are wash with buffer, then the secondary conjugated antibodies are pipeted in. Then they are washed again. Then the substrate is put in. The positive results will show the conjugated secondary antibodies.
E test
Where a plastic strip with a concentration gradient are impregnated on one side.
Yeast VS bacterial Cell
Yeast: budding, large, and nucleus and other internal structures Bacteria: small, no budding, no internal structures
Glucose, lactose, and manitol
Yellow means acid production and carbohydrate fermentation. Bubble means gas production.
parafocality
ability to remain in focus when switching objectives.
in the precipitin test, what is present in the serum, "agent" of disease
anti-BSA
white precipitate
anti-BSA + BSA, how do you know that antibody-antigen interaction has taken place
primary antibodies
antibodies present in tested serum samples as result of an individual's exposure to a pathogen
acidic dyes
are negatively charged and work best at low pH
Arm of microscope
backbone of the microscope
Hats should be worn _______
backwards
gram positive
bacteria that have a cell wall less complex and contains more peptidoglycan than the cell wall of gram-negative bacteria. (Gram-positive bacteria are usually less toxic than gram-negative bacteria. and stains purple)
acid fast stain
binds strongly only to bacteria that have a waxy material in their cell walls ( wax prevents stains from gram stain) Primary stain: carbolfuchsin Mordant: turgitol & phenol Decolorizer: acid-alcohol Counterstain: methylene blue (Mycobacterium tuberculosis, Nocardia sp.)
What are the disinfectants and antiseptics we used?
bleach, lysol, iodine, 70% ethanol, H2O2m, isopropyl alcohol
Concentration equation
c.f.u./(volume plated)x(total dilution)
What is halotolerant?
can survive high salt concentration but doesn't require them.
hypotonic
cell could burst from an excess buildup of osmotic pressure, most bacteria, more turgid, lower concentration of solutes exists inside the cell, water will be drawn into the cell
Maneval's stain
cell is red and capsule is not stained
3 media classification
chemically, functionally, nutritionally
TSA
complex, rich, general purpose
PIA
complex, rich, selective Pseudomonas Isolation Agar
MacConkey
complex, rich, selective differential
BHI
complex, very rich, general purpose Brain Heart Infusion
Is cheeze creating constructive or destructive
constructive
simple stains
crystal violet, used to determine bacterial size, morphology, and cellular arrangement
classification of media MM1, MM1 without glucose, MM1 without sulfur
defined, minimal, selective
Heterotrophic
depend on an outside food source
Is ground beef constructive or destructive, when not cooked
destructive
Catalase Test
drop H2O2 and see if there are any bubbles, if there are bubbles there is catalase activity.
Are endospores or vegetative cells more likely to be heat resistant?
endospores.
Size of S. cerevisiae
eukaryote 5-10 micrometer
Elevation of colony (6)
flat, Umbonate (tiny hump on convex surface), Raised, hilly (rough), craterform (concave with the sides raised up), convex.
Order of ELISA
fluid sample, secondary antibody, substrate
secondary antibodies
generated to react specifically with the antibodies in the initial Ab-Ag complexes and allow detection of these complexes at concentration where precipitation or agglutination reaction normally do not occur
basic dyes
have a positive charge which is attracted to negative charge of bacterial cells
places where biofilms are
hospitals, anywhere
what helps endospore with high temp resistance
hydrophobic proteins in the endospore coat to maintain the low internal concentration of water
intermediate
indicates that the antibiotic may be useful under certain circumstances (effective in high concentration etc)
resistance
indicates that the organism causing the infection is resistant to the antibiotic and the antibiotic may not be the appropriate choice to treat this particular infection
susceptible
indicates that the organism causing the infection is susceptible to this antibiotic and the antibiotic may be the appropriate choice to treat this particular infection
Photoautotroph
inorganic compound carbon source, light energy source
Chemoautotroph
inorganic coupound carbon source, inorganic compound energy carbon source
all used glassware contaminated or not
items that must be disposed in disposal cart
hypertonic
loss of water from bacterial cell can result in plasmolysis, death of bacterium, higher concentration of solutes exists outside the cell, water will be drawn out of the cell
neutrophile
majority of bacteria have a pH optimum ranging from pH 6.5-7.5
How can a lower temperature affect generation time?
make it longer.
gram negative
more complex cell wall and contains less peptidoglycan ( Gram-negative bacteria are often more toxic than gram-positive bacteria. (appear pink- lose purple in gram stain) )
Acid stains tend to be ___________ because the color is carried by the negative ion.
negative stains
Is autoclaved milk constructive or destructive
neither
isotonic
no net movement of water molecules, cell structure not affected, equal concentration of solutes exists outside and inside the cell
Chemoheterotroph
organic compound as carbon source, organic compound as energy source
Photoheterotroph
organic compound carbon source, Light as energy source
Oculars
part of microscope you look into
Stage clip
part that holds the microscope slide in place
What antibiotic is produced by polymyxa, What is it more susceptible to
polymixins, gram negatives.
Size of E. Coli
prokaryote .5-2 micrometer
used microbe slides, toothpick, pipets, centrifuge tubes, swabs, broken glassware
red biohazard container
parfocality
remains close in focus as it switches from objective to objective, minor adjustments with the fine focus knob may be necessary
obligate aerobes
require oxygen
SEM
scanning electron microscope shows 3D picture
Goal of using KIA
simultaneous use of glucose an lactose in the medium, Also be used to detect proteins included in the medium
planktonic cells
single celled microbes
when ELISA is used to determine if an individual has been exposed to a particular disease, it detects the presence of
the antibody whose production was triggered by the pathogen
Virulence
the level of difficulty of a certain pathogen to penetrate the hosts defenses.
minimal inhibitory concentration (MIC)
the lowest concentration of an antibiotic that is needed to inhibit the growth of a particular organism under lab conditions, use Etest
Why do you invert plates
to prevent excess condensation
TEM
transmission electron microscope Shows inside of cell
Consistency of a colony (3)
transparent, tranlucent (blocks some light), opaque
wrappers, uncontaminated paper towels, gauze
trash bin
Cytochrome oxidase activity test
use a toothpick to transfer a large amount of cells from each of the pure culture plate to filter paper, add oxidase reagent purple indicates positive result
Catalase activity test
use an inoculation loop to add a visible amount of cells from each of the pure culture plates to the microscope slide add one drop of 5% H2O2 (bubbles if positive) (Bacteria produces catalase, which breaks down toxic H2O2 from aerobic respiration into H2) and O2.)
differential stain
used to more than one dye to stain an organism and show cell components.
