Lab Practical #1 Flashcards
Aseptic
Without contamination
Inoculation
Transfer of bacteria or other organisms into or onto a suitable growth medium (media = lab food)
Differential medium
Snyder agar
Normal flora
Organisms that live in or on body surfaces that do not normally cause diseases
Transient flora
Organisms that colonize the body for short periods of time due to environmental exposure. Easily disrupted by hand washing, antibiotics, etc.
Resident flora
Well-established species that thrive in or on the body. Will re-establish after disruption
Pathogens
Organisms that cause disease
Opportunists
Organisms that are generally non-pathogenic, but will take the opportunity to do so under certain conditions
- after antibiotic therapy
- in the very young or old
- immunocompromised individuals
Nutrient agar (NAP)
- seaweed carbohydrate
- rhodophyta
- bacteria do not use agar as energy
- agar melts at 100* C, re solidifies at 45* C
Nutrient broth (NB)
- Sterile H2O
- yeast extract
- beef peptones
- NaCl
- pH buffers
TSA/B
Trypticase soy agar/ broth
Boiling water
100* C or 212* F
Freezing
0* C or 32* F
Body temp
37* C or 98.6* F
Room temp (RT)
25* C or 68-72* F
Aliquot
Measured amt
Meniscus
Curve of the liquid
Dental Caries Exp
Org. = Streptococcus mutans (GPC) - turns sucrose into sticky carb called dextran - colonization forms a biofilm on teeth - biofilm becomes "dental plaque" - leads to tooth decay and causes cavities, or "caries" Media used = Snyder agar - pH indicator: bromcresol green (4.8) - alkaline color = green - acid color = yellow
Isolated colonies
Visible areas of growth, distinct from other colonies
Pure culture
- subculturing isolated colonies
- only one type of bacteria growing in or on a culture medium
Quadrant Streak Plate Exp.
- made to separate individual organisms from a mixed culture Mix 1: - Eschrichia coli (GNR) - Micrococcus luteus (GPC) Mix 2: - Escherichia coli (GNR) - Serratia marscecens (GNR) - Staphylococcus epidermidis (GPC) NAP marked with an X and 1/2" streak 3 quadrants plus a lazy S for 4th quadrant
Magnification
Making an object appear larger
Resolution
Ability to see that 2 objects are separate
Brightfield Microscope Magnification
Of objective lens
(4x) red = scanning
(10x) yellow = low power
(40x) blue = high power
(100x) white = oil immersion
Total Magnification
Ocular X Objective
4x X 10x = 40x
10x X 10x = 100x
40x X 10x = 400x
100x X 10x = 1000x
Rheostat
Varies the amt of voltage of light obtained
- scales from 1-5 (dark to light)
Parfocal
Equal focus
Working distance
Between the objective lens and the slide
Depth of Field
How many layers you can see
Revolving Nosepiece
Houses the objective lenses
Condenser
Focuses all light to the stage
- raise the light, lower the contrast
- houses the iris diaphragm
Iris Diaphragm
Changes the amt of light going from the lamp to the stage.
Size of Field
Area of a slide being viewed through the ocular lens
- round circle of light
Refraction
Bending or scattering of light
Refractive Index
The degree that light bends
- a measure of the bending power of a medium
(Ex. Refractive index of immersion oil is the same as glass)
Steps to alter lighting of the microscope
A) adjust the iris diaphragm
B) move the condenser up or down
C) change the rheostat
Simple Unstained Wet Mount
- place one drop of liquid material in center of clean slide
- place cover slip @ 45* angle over slide and let go so it covers entire drop of liquid
- view slide in Brightfield microscope
Recite smear prep using both broth cultures and solid media
:)
Chromophore
Part of the dye that contains the color
Basic Dyes
- methylene blue
- malachite green
- crystal violet
- safranin
- basic dyes stain the cytoplasm of the cell
- *best for bacterial smear
- **positively charged chromophores stain negatively charged materials
Simple Stain
- make a heat fixed smear
- place slide on staining rack
- flood slide with methylene blue or other basic dye
- allow dye to sit for 1 min.
- rinse off excess dye with tap water
- allow slide to dry
- view!
Salt
Chemical compound made of two parts
- positively charged part (cation)
- negatively charged part (anion)
Attraction of the two components forms the ionic bond
Acidic Dye
(Ex. India ink)
- used in a negative stain
- used making a push smear
- negatively charged chromophores stain positively charged materials
- faster
- done in one step
- morphology is more accurate
- no heat fixing
Negative Stain
- one drop of India ink at one end of slide
- stir organism in drop of India ink
- make a push smear
Spirochetes
Free-living, non-pathogenic organisms that are very rare in microbiology
Gram Stain
-differential stain Primary stain: Crystal Violet (1 min) Mordant: Gram's Iodine (1 min) Decolorizer: 95% Ethyl Alcohol (3-5 sec) Counterstain: Safranin (2 min)
Blue cells = gram positive
Red cells = gram negative
colors the cell wall
**originally used to distinguish Streptococcus pneumoniae and Escherichia coli
**we tested Staphylococcus epidermidis and Escherichia coli
Target circles
Sharpie pen used to make a small circle in the center of the slide. This is where the organism will be placed onto the slide.
Subculture (sub)
Taking organisms from an original “primary plate” or broth and placing then onto or into another plate or broth
Problem: organisms aren’t blue enough
Reason 1: crystal violet not left on long enough
- increase timing of primary dye
Reason 2: iodine not left on long enough
- leave iodine on same amt of time as cv
Problem: gram + appear -
Reason: over decolorizing
- decrease time of ethyl alcohol
Problem: gram - appear +
Reason: under decolorizing
- either increase decolorizer time or make thinner smear
Problem: organisms not red enough to see
Reason: safranin not left on long enough
- increase timing of safranin
Problem: organisms stain unevenly due to state of cell wall
Reason: orgs are too old
- use fresh cultures (18-24 hours old)
Special stains
Used to see structures like flagella, endospores, and capsules
- acid-fast stain
- Shaeffer Fulton Endospore stain
- capsule stain
Acid-Fast Stain
-dual smear Primary Dye: carbolfuchsin (5-7 min) Decolorizer: acid alcohol (10-30 sec) Counter stain: methylene blue (2 min) -slide is steamed with bibulous paper and carbolfuchsin over tea light
AFB’s are bright fuchsia pink
Non-AFB’s are blue
Orgs. used: Mycobacterium smegmatis (AFB) and Micrococcus luteus (non-AFB)
Dual smear
Two bacterial species on one slide
AFB
Acid Fast Bacilli
Endospore Stain
-Schaeffer Fulton Endospore stain
Primary dye: malachite green (5 min)
Decolorizer: long rinse w tap water (30 sec)
Counterstain: safranin (2 min)
- steamed with bibulous paper and malachite green dye
Green endospores and red vegetative cells
Orgs used: Bacillus subtilis (old and young)
Endospores
Only made by a few genera of bacteria
-notably Bacillus and Clostridium
Sporulation
Spores are not fully extruded from the cells
Glycocalyx
Bacterial capsule
Capsule Stain
Primary dye: India ink (push smear)
Decolorizer/fixative: 0.1N HCl (30 sec)
Counterstain: safranin (1 min)
-make a push smear with the India ink, let air dry and flood smear with HCl. Then poor off excess and stain with safranin.
Red colored rods or yeast cells (vegetative cell = red)
Orgs used: Klebsiella pneumoniae, Bacillus megaterium, and Saccharomyces cerevisiae (yeast)
Prepared slides viewed in class
- Treponema pallidum
- syphilis
- spirochetes
- Neisseria gonorrhoeae
- gram - intercellular diplococci
- Flagella
Turbidity
Cloudiness
Counting Colonies (NAP)
0 colonies = NG 1-10 colonies = 1+ 11-50 colonies = 2+ 51-100 colonies = 3+ Over 100 colonies = 4+
Counting Colonies (NB)
No turbidity = NG Slightly turbid = 1+ Moderately turbid = 2+ Mod to heavy turbid = 3+ Heavy turbidity = 4+
CFU
Colony-forming units
- gram if solid material
- ml if liquid material
Nutritional requirements
Carbon: autotrophs or heterotrophs? Nitrogen Minerals Vitamins Growth factors: fastidious or not? Energy source: phototrophs or chemotrophs? Water
Physical requirements
Temperature: mesophilic, thermophilic, or psychrophilic?
pH: acidophilic or alkalophilic?
Atmosphere: aerobic or capneic?
Agar
- solidifying agent
- polysaccharide derived from algae
(Rhodophyta) - thickens liquid
- complex carb
- bacteria do not use as a nutritional source
Serial dilution
Series of repeated steps
Titer (concentration)
___ # CFU/ml or gram
Dilution
Amount transferred/ new total volume
Bacterial population counts
> 300 TNTC (too numerous to count)
<30 record #
Inbetween find the titer
Direct microscopic counts
- diluting out # of organisms and placing in calibrated fields on a slide
- # of orgs counted is X by dilution factor (DF)
- good for evaluating safety of milk
- counts dead and alive orgs
Turbidometric Methods
# of orgs vs spectrophotometer reading, then tubes are plotted on the standard curve - both dead and alive orgs
Standard Plate Counts (SPC)
- serial tube dilutions
- played into solid media
- DF on each plate used to determine CFU per ml
- only counts living orgs
Quebec Counter
Aids in counting colonies by magnifying and back-lighting the agar plates
Metabolism
- aerobic respiration (38 ATP)
- anaerobic respiration (32-34 ATP)
- fermentation (2 ATP)
(ATP/glucose molecule)
Thioglycollate Broth
- resazurin is the O2 indicator
Anaerobic Culture Ex 18
Orgs used: Alcaligenes faecalis, Staphylococcus aureus, Escherichia coli, Clostridium sporogenes
Media used: thioglycollate broth, NB, NA slant
-37* plate is the control
Capneic
Orgs that like CO2
Disinfectants
Decrease # of vegetative cells on an inanimate surface
Antiseptics
Decrease # of vegetative cells on living tissues such as skin
Chemotherapy
They are taken into the body to control infection
- antibiotics and synthetics
Antibiotics
Substance against life
- penicillin
Synthetics
Drugs that do not come from a living source
Broad spectrum
Chemical agent that works against both gram types
Narrow spectrum
Chemical agent that is effective against either gram + or gram - orgs
Sensitive vs Resistant
:)
Cidal
Totally killing organism
Static
Inhibition of growth of org
KB Test
Kirby Bauer Antibiotic Sensitivity Test
- standardized procedure to determine best antibiotic to use for treatment of a patient isolate
Agar Diffusion Method
Saturating paper discs with agent and allowing the discs to incubate on the surface of agar plates previously inoculated with the test organism
Zone of Inhibition
Clear area of “no growth” around a specific disc
Chemical Control Agents Ex 21
Orgs used: Staphylococcus aureus, Escherichia coli, and Bacillus subtilis endospores
ATCC
American Type Culture Collection
- where we get our cultures for lab
Factors Standardized for KB Test
- Swab technique (confluent growth)
- Discs- size, spacing, pre-labeled, pre-saturated
- Mueller Hinton agar plate- plate size, depth= 4mm, pH= 7.2-7.4
- Temp.= 35+-2*C
- # of orgs- MacFarland standards 0.5
- Age of orgs- 18-24 hours
- Incubated 16-18 hours
- QC orgs
KB Test Ex 22
Orgs used: E. Coli, Staph. aureus, enterococcus faecalis, Serratia marcescens, Pseudomonas aeruginosa, Baccilus megaterium
- 8 pre-saturated discs of antibiotics
