MCB 2610 Study Guide - Final Guide: Trypticase Soy Agar, Crystal Violet, Bunsen Burner
25 May 2018
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Microbiology Lab Basics:
T-streak= aseptic + colony morphology
Aseptic ensures that any chance of picking up contaminating microbe is minimized
(quiz question: why is aseptic technique important)
Isolation of Bacteria :
We isolated two species from a mixed culture sample and established two pure cultures with
tryptic soy agar plates
Streak Plate Method- to isolate bacteria by spreading it out on the agar surface of a petri dish.
3 Phase Streak Method: after isolation, the first section that was streaked had the heaviest
growth, and the other sections showed isolated colonies.
We then chose a isolated colony and then restreaked that colony on a second plate and it was
incubated.
Distribution of Microorganisms:
5 different nutrient agar (NA) plates:
1. 30 min exposed to air
2. Jenn’s hair over the dish
3. Swab of not disinfected bench
4. The floor
5. Cici teeth
We observed all the agar plates and the number of colonies and described it. PAGE 26
Form: punctiform, circular, filamentous, irregular
Elevation: flat, raised, convex
Margin: entire, undulate.
Use of the Optic Microscope:
PAGE 30
You only use the immersion oil when working on the 100X objective lens.
Preparation of Slides for Staining:
Staining procedures that only use one stain are called simple stain and this helps to determine
cell morphology, size and arrangement.
- Direct stain: a simple stain that colors the bacteria
- Negative stain: stains the background but leaves the bacteria unstained
We drew circles with the wax pencil, then we grabbed a culture with the loop and then added a
drop of water in the circle (allowing it to air dry). Put over flame (heat fix denatures bacterial
enzyme and it enhances the adherence of bacterial cell to microscope slide). We did a simple
crystal violet stain.
Differential Staining:
Stain: synthetic aniline dyes derived from benzene : methylene blue chloride → methylene blue
(chromophore) + Cl-
Most bacteria are stained when a basic stain permeates the cell wall and adheres by weak ionic
bonds to the negative charges of the bacterial cell.
Gram Staining:
Crystal violet solution (1 min) → iodine → ethanol wash (removes the crystal violet dye) →
Safranin
Allows us to classify the bacteria into two categories based on cell envelope structure:
●Gram positive: bacteria that retains the stain
○Thick mesh like cell wall made of peptidoglycan that retains the purple stain
○1 inner layer and peptidoglycan layer
●Gram negative: does not retain the stain
○Outer membrane and a thinner layer of peptidoglycan that is easily discolored and
only retains the counterstain (safranin pink)
○Outer & inner layer & peptidoglycan
PAGE 40 ex. of microscope
Spore Staining:
Bacillus
and Clostridium
develop an endospore in response to certain environmental conditions
such as depletion of nutrients (in our case it was carbon).
** Heat used during staining does NOT cause sporalation.
We cut pieces of bibulous paper over the smears and placed the slides over a tripod with the
bunsen burner underneath. We flooded the paper with 5% aqueous malachite green dye.
From assignment questions:
1. Why is differential staining important
2. Under what conditions does sporulation form
Motility:
Brownian Motion: erratic movement; random collisions
True Motion: Quick movement in straight/curved lines
Motility Agar:
●Tubes containing non-motile bacteria will be only red along the stab path
●Bacteria that are motile will have red distance from the stab path and are capable of
growing in areas of agar which have lower oxygen levels.
●If the culture is an aerobe, then it will grow at the surface of the tube and only slightly
away from the stab line.
Page 74 for example of pictures
Microbial Growth:
Defined as an increase in population size.
Growth curve: lag phase, log phase, stationary phase, and death phase.
●Lag Phase- occurs when an inoculum (bacteria) is transferred to a fresh growth medium,
they are adjusting to the new conditions and synthesize a full complement of enzymes.
○Cell is getting ready for division
●Log Phase- cells divide, grow and divide again!
●Stationary Phase- when the culture is in a closed vessel it exhausts the limiting nutrients
that is available for growth and so the division of cells slows.
○it is a flat line because cell dividing and cell dying are happening at the same time
and they cancel each other out
●Death phase- caused by the accumulation eventually of toxic waste products, by cell
damage from exposure to light or chemicals in the environment.
In lab, we measured the growth rate of E. coli
at 37 and 28 degrees celsius.
The generation time was SHORTER at 37 degrees celsius meaning this was the optimal
temperature for growth of the organism. (higher temp speeds up growth rate)
Gram Positive Cocci:
Normal Flora/ Indigenous: microorganisms found in many regions of the body
Fomites: can transmit pathogenic strains of staphylococcus between individuals.
Mannitol salt agar: S. Aureus
will ferment mannitol to produce acid so mannitol sugar agar is
used to select and differentiate
- When fermented the acid produced as a by-product turns the plate yellow
- Pathogenic strains of S. aureus
produce the enzyme coagulase
Streptococcus pyogenes are pathogenic (bacteria in throat) and gram positive!!
Streptococcal species produce enzymes called streptolysins which cause cell lysis
Hemolysis: blood cells under attack that lyse
Hemolysis can be determined by streaking bacteria on plates of blood agar.
●Alpha hemolysis- partial hemolysis of rbc’s.
○Characterized by a green/cloudy zone around the bacterial growth
○Streptococcus pneumoniae and streptococcus sanguis do we have to know this??
○Green=partial
●Beta hemolysis- complete hemolysis
Document Summary
Aseptic ensures that any chance of picking up contaminating microbe is minimized (quiz question: why is aseptic technique important) We isolated two species from a mixed culture sample and established two pure cultures with tryptic soy agar plates. Streak plate method- to isolate bacteria by spreading it out on the agar surface of a petri dish. 3 phase streak method: after isolation, the first section that was streaked had the heaviest growth, and the other sections showed isolated colonies. We then chose a isolated colony and then restreaked that colony on a second plate and it was incubated. 5 different nutrient agar (na) plates: 30 min exposed to air, jenn"s hair over the dish, swab of not disinfected bench, the floor, cici teeth. We observed all the agar plates and the number of colonies and described it. You only use the immersion oil when working on the 100x objective lens.
