Bio 2025 Lab Practical 1
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Know the difference between Normal and Transient flora.
- Normal flora : Resident Bacteria “good” bacteria. ex: Staph
- Transient Flora : Pathogenic bacteria.
- ex: Salmonella
General procedures/techniques for aseptic transfers
- Label, flame inoculation tools.
- Flame lip of the tube : minimize the production of aerosols.
Purpose for using aseptic techniques.
- Minimizes contamination/decrease the commination of the culture.
- Decrease the contamination of students.
- And the environment.
Purpose for sub-culturing bacteria
- Done because food/space runs out
- Waste products from the bacteria start accumulating.
Differences between Broth, Slant and Plate medias.
- Broth : used to grow microbes when fresh cultures or large numbers of cell are required.
- Agar slants : used to grow stock cultures that can be refrigerated after incubation and maintained for several weeks.
- Plate : Used for obtaining isolation of species, differential testing, and quantifying bacterial densities.
Why do we streak for isolation?
- Pure culture contains only a single species.
- Mixed culture contains two or more species.
- We streak for isolation to find the individual species from a mixed sample.
The dyes used for the simple stain and purpose of a simple stain.
- Crystal Violet, Safranin, Methylene Blue
- Applies to bacterial smears that have been heat-fixed. Colors the cells and makes them more visible under the microscope. Cell morphology, size and arrangement can be determined.
Common bacterial cell morphology
Common bacterial cell arrangement
Steps when perfuming a Gram Stains.
What does each dye (CV, Iodine, Safranin) do?
What does the decolorizer (ethanol) do?
Purpose of a gram stain.
How do the cells look after each step?
color of G+ vs G- cells when staining is done correctly
color of G+ vs G- cells when staining is done incorrectly
Draw it out and answer!
- 1. Primary stain is crystal Violet. Iodine is added to enhance the crystal violet by binding to the crystal violet and makes the molecule larger. Decolorization with an alcohol solution (most critical step) causes the peptidoglycan to shrink; Causes the Gram-negative cells to be decolorized and Gram-positive cells are not. Counterstain safranin is added and gram-negative cells can appear reddish-pink while Gram positive cells appear purple.
- Purpose : to distinguish between gram-positive and gram-negatve cells.
- Refer to pg. 169 Lab Manual
Purpose of the negative stain (why don’t we heat fix?)
- Used to determine morphology and cellular arrangement in bacteria too delicate to withstand heat-fixing. Also produces minimal cell shrinkage.
- Example : Treponema (becomes distorted by heat-fixing.)
Purpose of heat fixing our cultures
- Allows the bacteria to latch onto the slide.
Correct labeling of test tubes
- Test Tubes : Name, Date and Organism.
- Inoculation : Name, Date, Medium, Organism and Table #
What are the general procedures for an ACID FAST stain and what’s the resulting colors?
- - Primary Stain : Carbol Fushion
- - Decolorizer : Acid Alcohol
- - Secondary Stain : methylene blue.
- - Results :
- (+) = red/pink
- (-) = blue
What’s the purpose of using heat + steam during the Acid Fast staining procedure?
- Steam loosens the waxy layer (lipoidal layer made of mycolic acid) allowing stain to penetrate.
What's the purpose of the Acid Fast Stain?
- Checks for the presence of mycolic acids, a thick lipoid layer characteristic of a mycobacterium.
Why do we use acid alcohol to decolorize instead of ethanol/Gram’s alcohol?
- Regular alcohol is not strong enough to decolorize.
In a hospital setting, the Acid Fast stain can be used to diagnose which disease?
- Tuberculosis which is caused by mycobacterium Tuberculosis.
What is the function of mycolic acid?
- Used by mycobacterium to evade/hide from out immune system.
Know the general procedure for the SPORE stain and resulting colors.
- - Primary Stain : Malachite green (stains spore)
- - Decolorizer : H2O
- - Counter Stain : Safranin
- - Results :
- (+) = Green Spores
- (-) = Pink background/cell
What's the function of a spore and what’s it made of?
- - Dormant form of bacteria, allows to withstand harsh environments.
- - Made out of protein, Keratin.
Why do we use heat + steam in the spore stain?
- to allow the malachite green to penetrate the keratin outer layer and stain the spore.
What's the difference between the vegetative and spore forms of a bacterial cell?
- Vegetative : reproductive form
- Spores : dormant form.
Which genera produces spores?
- Bacillus and Clostridium
What are the different types of spores?
- - Location : central, subterminal, terminal
- - Shape : spherical, elliptical
Know the general procedure for a capsule stain.
- - Primary Stain : crystal violet
- - Decolorizer : copper sulfate
- - Counter Stain : NONE.
Function and composition of a capsule?
- - Increases virulence, protects from phagocytosis.
- - Capsules are made of polysaccharides
Why do we NOT heat fix for the capsule stain?
- Distorts and destroys morphology of capsules.
know the parts of the bacterial growth curve and what each indicates.
- Decline Phase
Know the general differences between the direct and indirect enumeration methods.
- Indirect method : (Spectrophotometer) counts both dead and alive cells.
- Direct method : (Serial dilutions + spread plate) counts only living cells.
Which bacteria would be considered so dangerous? Why?
- Bacillus Anthracis
- - It can form spores and capsules!
What’s the definition for CFU?
What’s considered a countable plate?
How do you calculate CFU/ml given a set of plates?
- Colony forming units
- 30 ~ 300
- Average # of Colonies x Dilution x Volume plated.
- Exponents of the dilution and volume plated are POSITIVE for the equation since we would have more bacterial cells without the dilution.
What are the types of environments created by the TORBAL JAR?
What type of bacteria can grow on each?
- Torbal jar creates an anaerobic environment.
- Candle: Microaerophile
Why do many of the bacteria that cause human disease are mesophiles?
- Mesophiles (20-45*C) and have an optimal growing temperature near body temperature at ~37*C.
Identify the type of environment where a bacteria grows best and the range.
- Temperature :
- - Psychophiles(trophs) / -5 - 30*C / P. Flourescens
- - Mesophiles / 20 - 45*C / E. Coli and Staphs
- - Thermophiles / 45 + *C / Geo. Stearothermophilus
- pH :
- - Acidophiles / 0 - 5.5 / Lactobacillus Plantarum
- - Neutrophiles / 5.5 - 8.5 / E.coli and Staphylococcus saprophyticus
- - Alkalophiles / 8.5 - 14 / Alcaligenes Faecalis
- Osmolarity :
- - Halophobes / 0-5% / E. Coli and Staphs
- - Halotolerant / 2.5 - 15% / Vibrio
- - Halophiles / 15% + / Halobacterium
- O2 requirements :
- - Aerobe / Needs O2 / Alcaligenes Faecalis
- - Micro Aerophiles / Needs some O2 / Lactobacillus Plantarum
- - Facultative / With or w/o O2 / E. Coli, Staphs.
- - Anaerob : NO O2 / Clostridium Sporogenes.
How do antibiotics work? What do they target?
- Prevents/interfere with DNA/RNA Synthesis.
- Prevents/interferes with protein synthesis.
- Disrupts cell wall/membrane formation.
Explain in detail the methods for antibiotic resistance?
- Altered target, usually caused by mutations. Antibiotics unable to recognize targets.
- Poor uptake, cell unable to take in antibiotic.
- Chemical inactivation (think B-Lactase.)
Explain how Augmentation works
The antibiotic augmentation is a mixture of amoxicillin (semi-synthetic penicillin) and clavulanic acid. Bacteria have become resistant to penicillin by producing B-lactamase which chemically inactivates the antibiotic. The addition of Clavulaic acid is able to deactivate B-lactamase making the antibiotic effective again.
What's bacteriostatic and bactericidal.
- Bacteriostatic : inhibits growth
- Bactericidal : kills bacteria
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