[BIO 120.12] Final Exam - Exercises 1 to 2

To obtain a pure or axenic culture of a microorganism

Goal of isolation?

TRUE

T/F: A pure culture is derived from the growth of a single species and consists of one type of cell

FALSE

T/F: A pure culture is derived from the growth of a single species and can contain more than one type of cell for as long as it is from one species

TRUE

T/F: Reactions of mixed cultures in characterization tests cannot be used for identification

FALSE

T/F: Reactions of pure cultures in characterization tests cannot be used for identification

characterization tests

FILL: Reactions of mixed cultures in [___ tests] cannot be used for identification

identification

FILL: Reactions of mixed cultures in characterization tests cannot be used for [___]

Uniform colony characteristics (on plates or tubes), Uniform cell morphology, Uniform Gram staining reaction

3 Criteria for Bacterial Purity

Spread Plating

Technique in Bacterial Isolation: Desired microorganism is usually present at higher level than any other microorganism

FALSE

T/F: Spread plating can only be used in isolation, but not enumeration

TRUE

T/F: Spread plating can be used in both isolation and enumeration

Isolation, Enumeration

What two techniques can be done with spread plating?

Spread plating, Streak plating

Two bacterial isolation techniques that depend on spatial separation of cells

Spread Plating

Bacterial isolation technique wherein low density mixed cells are spread out on an agar surface

FALSE

T/F: Spread plating involves having low density pure culture cells that are spread out on an agar surface

TRUE

T/F: Spread plating involves having low density mixed cells that are spread out on an agar surface

Dry Plates

In spread plates, what kind of plates must be used to prevent colonies from coalescing

TRUE

T/F: Spread plating may require serial dilution

FALSE

T/F: Streak plating may require serial dilution

Series of dilution will thin out microbial population until there is only one cell in a tube of diluent

What is the principle behind serial dilution?

Streak plating

Main method for isolation

Streak Plating

Bacterial isolation technique that relies on the chance that a single cell will drop form the loop as it is rubbed along the agar surface

Viability, Purity, True-to-Type (prevent mutations)

What are the three reasons why we must preserve bacterial cultures?

OK SSP SiRoA

6 Examples of Techniques in Bacterial Isolation

OK PO dry liquid low

6 Methods for Bacterial Preservation

Culture Characteristics

Appearance or growth characteristics of a particular culture on a medium

Culture medium

Characteristics of a bacterial colony may vary depending on the ___ used

OK STEM F BAS SiCo

10 Indicators of Purity But Not Typically Useful in Identification

General Purpose Medium

Nutrient Agar or broth is what kind of medium?

Nutrient Agar or Broth

Media that supports almost all microbial growth due to the lack of inhibitory substances

Accumulation of condensate on the lid is prone to contamination, Ease of handling and safety

Why do we cool the molten medium to 45^oC before dispensing into sterile petri dishes?

Moisture on the lid will drop on the surface of the agar, Colonies will coalesce (no isolated colonies)

Why are inoculated agar plates incubated in inverted position?

To get well-isolated colonies, Provide optimal growth conditions for aerobic bacteria

Why do we need to keep the inoculating loop from cutting into the agar surface?

Filiform, Echinulate, Beaded, Effuse, Aborescent, Rhizoid

Label the following growths on agar slant/stroke

Ring, Pellicle, Flocculent, Membranous

Label the following growths on broth

OK FE BEAR

What are the 6 different lines of punctures we can see for growth on stab?

0.85% saline

What was used in Experiment 1 to make the serial dilutions of the bacterial cultures?

121 deg C, 15 psi for 15 min

What are the sterilization conditions in an autoclave? Temperature, pressure, time

37 deg C

What was the temperature for incubation in Exercise 1?

45 deg C

What was the temperature in which the sterile NA was first cooled to before pouring?

Surface, Subsurface, Sedimentation

What are 3 things we look for in broth growth?

OK FlaTu FloGra

What are the 4 subsurface appearance in broth growth?

OK PeMe FloRi

What are the 4 surface appearance in broth growth?

Filamentous, Coccus, Coccobacillus, Vibrio, Bacillus, Spirillum, Spirochete, Fruiting

Name the Shapes of the Prokaryotic Cells

Stalked, Square, Star, Pleiomorphic

Name the Shapes of the Prokaryotic Cells

Diplococcus, Tetrads, Sarcinae, Staphylococcus, Streptococcus, Single, Diplobacillus, Streptobacillus, Rosette

Name the Prokaryotic Cell Arrangements

Staining

Coloring the microorganism with a dye that emphasizes certain structures

Makes observation easier, Specimen preservation, Makes specimens not infectious

What are the 3 advantages of staining?

Complex and tedious, More expensive

What are the 2 disadvantages of staining?

Smear Preparation → Fixation → Staining

What are the 3 general steps in staining?

TRUE

T/F: Gram staining is fundamental to the phenotypic characterization of bacteria

FALSE

T/F: Gram staining is fundamental to the phenotypic identification of bacteria

TRUE

T/F: Gram staining is used only for differentiating organisms in Domain Bacteria

FALSE

T/F: Gram staining is used for both Domain Bacteria and Archaea

Aids in antibiotic selection (narrow vs. wide spectrum antibiotics)

What is the clinical benefit of Gram Staining?

Narrow Spectrum Antibiotics

Antibiotics that are active against a selected group of bacterial types. They can act on either gram positive or gram negative but not both.

Wide Spectrum Antibiotics

Antibiotics that act on the two major bacterial groups, Gram-positive and Gram-negative, or any antibiotic that acts against a wide range of disease-causing bacteria

Thick peptidoglycan layer, Low lipid content

Describe Gram Positives in terms of: Peptidoglycan Layer, Lipid Content

Thin peptidoglycan layer, High lipid content

Describe Gram Negatives in terms of: Peptidoglycan Layer, Lipid Content

Heat Fixation → Crystal Violet for 1 Minute → Drain and Rinse with Distilled Water → Iodine Treatment for 1 minute twice → Drain and Rinse with Distilled Water → Tilt slide as you decolorize with 95% Ethanol for 10-15 seconds → Drain and Rinse with Distilled Water → Safranin for 1 minute → Drain and Rinse with Distilled Water → Observe under OIO

What are the specific steps for Gram Staining (from heat fix to OIO)?

CV+ and Cl- ions

In aqueous solutions, crystal violet dissociates into what kind of ions?

TRUE

T/F: CV+ ions can penetrate through the wall and membrane of both gram positive and gram negative cells

FALSE

T/F: CV+ ions can penetrate through the cell wall only, but for both gram positive and gram negative cells

FALSE

T/F: CV+ ions can penetrate through the wall and membrane of gram positives only

FALSE

T/F: CV+ ions can penetrate through the wall and membrane of gram positives, but only the membrane of gram negatives

It interacts with negatively charged components of bacterial cells

How does CV+ color the cells purple?

Mordant

a substance, typically an inorganic oxide, that combines with a dye or stain and thereby fixes it in a material

FALSE

T/F: A mordant is typically an organic substance that can combine and fix dye into a material

TRUE

T/F: A mordant is typically an inorganic substance that can combine and fix dye into a material

Mordant

Stain affinity enhancer

Iodine ions (I- or I3-) interact with CV+ to form large and insoluble CV-I complexes within the cytoplasm and outer layers of the cells

What is the rationale behind the usage of a gram iodine mordant?

TRUE

T/F: In the mordant step, iodine ions (I- or I3-) interact with CV+ to form large and insoluble CV-I complexes within the cytoplasm and outer layers of the cells

FALSE

T/F: In the mordant step, iodine ions (I- or I3-) interact with CV+ to form small but insoluble CV-I complexes within the cytoplasm and outer layers of the cells

FALSE

T/F: In the mordant step, iodine ions (I- or I3-) interact with CV+ to form large and soluble CV-I complexes within the cytoplasm and outer layers of the cells

FALSE

T/F: In the mordant step, iodine ions (I- or I3-) interact with CV+ to form large and insoluble CV-I complexes within the outer layers of the cell only

Use of 95% ethanol as decolorizer

What is the most critical part in gram staining

Differentiates gram positive from gram negative cells

Why is the usage of 95% ethanol (decolorizer) the most critical step in gram staining?

Removes dye from stained cells

What does the 95% Ethanol do in Gram staining?

Gram Positive

Gram Positive or Negative: Low lipid extraction

Gram Positive

Gram Positive or Negative: Pores are shrunk

Gram Positive

Gram Positive or Negative: Decreased permeability due to dehydration of multilayered peptidoglycan

Gram Negative

Gram Positive or Negative: High lipid extraction

Gram Negative

Gram Positive or Negative: Increased porosity and leaky cell walls

Gram Negative

Gram Positive or Negative: Outer membrane is lost from cell

Gram Negative

Gram Positive or Negative: Peptidoglycan layer is exposed

Gram Positive

Gram Positive or Negative: CV-I complex cannot pass through

Gram Negative

Gram Positive or Negative: CV-I complex becomes washed off from cells and they become colorless

Negative charges of the cell have already interacted with CV

In Gram positives, why do their cells remain purple even after the safranin counterstain step?

Negative charges remain free so the cells can take up the counterstain

In Gram negatives, why do their cells get colored after the safranin step, unlike gram positives?

Older cultures can have breaks in cell wall and often give gram variable results

Why is the age of bacterial culture an important consideration in gram staining?

Thickness of smear (number of cells on the slide)

What determines the degree of decolorizing required in gram staining?

Over-decolorizing

This will lead to an erroneous result where Gram-positive cells may stain pink to red indicating a Gram-negative result.

To prevent gram positive cells from staining pink

Why do we keep the slide tilted during the decolorization step?

Under-decolorizing

This will lead to an erroneous result where Gram-negative cells may appear blue to purple indicating a Gram-positive result

Gram variability, Cell walls become particularly sensitive to breakage during cell division, Decrease in peptidoglycan thickness during growth

Under nature of organism, what are 3 factors that may affect gram reaction?

Actinomyces, Arthrobacter, Corynebacterium

3 examples of bacteria with Gram positive structures but may stain pink to red as their cell walls are sensitive to breakage during cell division

Bacillus, Butyrivibrio, Clostridium

3 examples of bacteria who are gram positives but may stain pink or red due to decrease in peptidoglycan thickness during growth

Age of bacterial culture, Thickness of smear, Nature of organism

3 factors affecting gram reaction