Lab practical 1 Flashcards
What is appropriate PPE?
Gloves, lab coats, closed toe shoes, long hair tied
Where do gloves and used plate cultures go for disposal?
Biohazard bag
Moves stage up/down and side to side
Stage controls
Moves stage closer/further from object lens
Course focus
Brings specimens into sharp focus
Fine focus
Focuses light
Condenser
Varies the intensity and size of the cone of light
Iris
Total magnification of light compound microscope =
Objective lens x eyepiece
Used to describe the sharpness and detail of the image
Resolution
Used to describe the enlargement of an object under the microscope
Magnification
Sphere microorganisms
Coccus
Rod microorganisms
Bacillus
Spiral microorganisms
Spirillum
Chain microorganisms
Strepto
Cluster microorganisms
Staphylo
Types of microscopy: light goes through the condenser
Dark object on light background
Bright field microscopy
Types of microscopy: light object on a dark background
Unstained organisms
Dark field microscopy
Types of microscopy: hollow cone of light
Improves contrast between object/background
Phase control microscopy
Types of microscopy: bright object dark background
Chemiluminescence
Phosphorescence
Fluorescence microscopy
Types of microscopy: electrons bounce off specimen
Visualization of the surface of specimen
Specimens coated in electron dense material
Scanning electron miscroscopy
Types of microscopy: beam of electrons pass through the specimen
Transmission electron microscopy
Multicellular fungi
Hyphae
Multicellular filamentous molds
Portion that obtains nutrients
Vegetative hyphae
Projects above the surface of the fungi, reproduction
Aerial hyphae
Incomplete budding of yeast cell
Pseudohyphae
Eukaryotes: fungi
Grow best at room temp and acidic ph 5.6
Fruiting bodies visible above ground
Macroscopic filamentous fungi
Eukaryotes: fungi
Single-celled
Multiple through budding
Sometimes opportunistic pathogens
Bread, brewing
Yeasts
Eukaryotes: algae
Unicellular, green
Photosynthetic
Found in temperature soils
Chlamydomonas
More closely related to eukaryotes than bacteria
Not pathogenic
Minority of exteemophiles
Archaea
Bacteria motility: corkscrew motion
Spirochetes
Bacteria motility: “run” and “tumble”
Flagellar
Bacteria motility: twitching/gliding motion
Pilli
Bacteria non-motile movement
Random
Collisions
Brownian movement
Staining: penetrates cell wall
Cell wall negatively charged
Positive charged any basic can be used (crystal violet, methylene blue)
Simple staining
Staining: stains background, not cell
Acidic dye, negatively charged
Negative staining
Staining: allows to differential between cell types
Gram staining
Differential staining
Gram stain: Purple stain
Gram positive
Gram stain: pink stain
Gram negative
Two extremes: oxygen is 100% intolerable, oxygen is 100% required
Obligate anaerobe or aerobe
Oxygen not necessary but can be tolerated
Oxygen may be beneficial
Facultative anaerobe
Cannot use oxygen
Oxygen not harmful
Aerotolerant anaerobes
Need oxygen but at lower levels
Microaerophiles
Kills microbes from tools, media, tables, other surfaces, gloves, etc.
Sterilization
Methods of sterilization
Autoclaves (moist heat)
Oven (dry heat)
Filtration
Radiation (UV and gamma)
Types of media: allows for fast growth
High density of microbes
Liquid (broth)
Types of media: used to check for motility
Anaerobic growth
Semi-solid
Types of media: used for colony isolation
Solid (agar)
Any broth can be made solid
Types of media: selects for certain types of bacteria
Selective media
Types of media: differentiates between bacteria
Differential media
Plating techniques: plate liquid sample containing bacteria
Can determine amount of bacterial cells in original liquid culture
Spread plate
Micropipette ranges: 0.1-2.0
P2
Micropipette ranges: 0.5-20.0
P20
Micropipette ranges: 20.0-200.0
P200
Micropipette ranges: 100-1000
P1000
Counts love and dead to get total number
Direct counting
Only counts living
Viable counting
Viable counting range
(TFTC) 30-300 (TNTC)
Light is inversely proportional to number of cells
Spectrophotometer
Turbidity
Dilute culture in stepwise manner
Serial dilution
Measures light absorbance at specific wavelength
Spectrophotometer
Phases of bacterial growth: No cell division
Lag phase
Phases of bacterial growth: generation time constant in
Exponential growth phase
Phases of bacterial growth: reproduction and death are balanced in
Stationary phase
Phases of bacterial growth: death rate exceeds reproduction in
Death phase
Common storage methods for culture samples
Agar slants
Permanents
Freezing cultures
Lyophilized cultures
Recommended storage time: less than 2 weeks
Agar plates
Recommended storage time: 3weeks to 6 months
Agar slants
Recommended storage time: 3 weeks to 12 months
Stored at room temp or cooler
Stab culture
Long term storage 1-3 years
-20C or -80C
Freezing cultures
Long term storage 15+ years
Culture is dried and frozen
Lyophilization
Common stressors
Temperature
pH
Osmotic pressure
Radiation exposure
Temperature classifications: 0-20C
Psychrophiles
Temperature classifications: 20-45C
Mesophiles
Temperature classifications: 45-80C
Thermophiles
Temperature classifications: 75-130C
Hyperthermophiles
pH classifications: pH 0 to 5.5
Vinegar, lemon juice
Acidophiles
pH classifications: pH 5.5 to 8
Water, blood
Neutrophiles
pH classifications: pH 8 to 14
Bleach, drain cleaner
Alkalphiles
Osmotic pressure classifications: low (solute) outside cell compared to inside
Hypotonic
Osmotic pressure classifications: high (solute) outside cell compared to inside
Hypertonic
Osmotic pressure classifications: equal (solute) inside/outside
Isotonic
DNA repair: need light for activation
Photolyase
Repairs thymine dimers
Less likely to cause mutations
Photoreactivation
DNA repair: recombination repair
DNA polymerase repairs new gap
Prone to mutations
RecA
Several different species occupying the same area
Community
Amplifies known sequence of DNA via ‘primers’
Applications: sequencing, molecular cloning, forensics, diagnostics (COVID)
Polymerase chain reaction
Derived from a thermophilic bacterium
Thermostable up to 95C
Taq polymerase
Utilizes temperatures and duration that are programmed into the machine
Thermocycler
Control that isn’t expected to give result
Negative control
Control that is expected to give result
Positive control
Highly concerved
All microbes will have the same NT sequence
Positive control
fis
Variable gene
Different microbes have different NT sequence for this gene
Allow us to identify the organism
dps
Relative ability of microbes to survive and grow in a particular environment
Fitness
Can evolve to increase their fitness:
Horizontal gene transfer
Recombination
Mutation
Separates nucleic acids/proteins based on size
Molecules are mobilized in an electric field - RUN TO RED
Gel electrophoresis
Made of agarose and buffer
Increased % is better for smaller molecules
Decreased % is better for larger molecules
Agarose gel
Adds weight to the DNA to keep it in the well
Easier to see
Loading dye
Contains fragments of known sizes
DNA ladder
Binds to DNA and allows visualization once placed on blue light
Gel stain
Soil Horizons: O =
Humus layer
Soil Horizons: A =
Topsoil
Soil Horizons: B =
Subsoil
Soil Horizons: C =
Regolith
Soil Horizons: R=
Unweathered bedrock
What have the largest biomass
Fungi
What produce around 75% of natural antibiotics
Actinomycetes
Secondary metabolites
Antibiotics
Antibiotics are not effective against
Fungi
The buildup and breakdown of nutrients within a cell
Reactions catalyzed by enzymes
Metabolism
Binding sites specific for substrate
Typically proteins
Enzymes
Biochemical reaction: Breakdown of large molecules that not smaller, more simple molecules
Makes energy
Catabolic
Biochemical reaction: takes smaller molecules and builds them into larger molecules
Uses energy
Anabolic
Building blocs of proteins
Some are essential and must be obtained externally
Amino acids
Organic molecules that contain carbon, hydrogen, oxygen, and nitrogen
Inactive without the correct shape
Proteins
Protein structure: a chain of AAs (polypeptide chain)
Primary structure
Protein structure: repetitive twisting/folding of the polypeptide chain
Creates two different structures: alpha helixes or beta sheets
Secondary structure
Protein structure: alpha helices or beta sheets fold into a 3D structure
Tertiary structure
Protein structure: multiple, folded polypeptide chains coming together to form a protein
Quaternary structure
SIMs test: production of hydrogen sulfide
Sulfur
SIMs test: hydrolysis of tryptophan
Indole
SIMs test: motile bacteria
Motility
IMVic test: hydrolysis of tryptophan
Indole
IMVic test: organic acid production
Methyl red
IMVic test: presence of acetoin
Voges proskauer
IMVic test: citrate as a carbon source
Citrate
Metabolic test: Multiple sugars, fermentation
Glucose, lactose, fructose
Sugar fermentation test
Metabolic test: indicates if microbe can ferment lactose
Beta-galactosidase
Metabolic test: breakdown of starch to glucose
Carbon source
Starch hydrolysis
Test the microbe’s ability to complete sulfur reaction
Most useful for determining presence/absence of enteric bacteria within a culture
SIMs test
Test if the microbe can utilize tryptophan as a carbon and nitrogen source vi tryptophase
SIMs test
Semisolid media used in this test allows for the identification of motile bacteria
SIMs test
Can only be used on gram negative bacteria
Determines if the microbe can use citrate as a carbon source
IMViC test
What are the types of microscopy
Bright field
Dark field
Phase control
SEM
TEM
Parts of the compound light microscope
Eyepiece
Observation tube
Neck
Coaxial stage controls
Course focus
Fine focus
Nosepiece
Objective lens
Condenser
Iris
Light source
What tool do you use to measure microbes
Spread plate or pipette?
What are the types of microbial motility
Flagellar
Pilli
Spirochetes
Why do we stack bacteria
Helps with differentiating microorganisms
What is a smear preparation
Spreading bacteria across a glass slide
What is heat fixing
Toughens structures for staining and inactivates enzymes
Compare and contrast simple staining to differential staining
Simple: only one stain used
Differential: multiple stains used
What are the three domains of life
Eukarya
Bacteria
Archaea
CFU/ml =
Number of colonies per ml plated / total dilution factor
What are thermoduric bacteria
Heat resistant bacteria that can survive pasteurization
Can tolerate high concentrations of salt
Halotolerant
How can UV radiation affect microbes
Stop them from repairing themselvez
What enzymes repair DNA damage
DNA polymerase
What is the SOS response and when does it occur
Stops DNA synthesis when damage is too great
What are taxa
Communities that consist of two or more different species
What does does PCR stand for
Polymerase chain reaction
Why do we use PCR
Able to obtain billions of copies of DNA quickly
What produces antibiotics
Soil microorganisms
