Microlab Final

KMS

Osmosis

The movement of water molecules through a selectively permeable membrane from an area of lower solute concentration to an area of higher solute concentration, aiming to equalize concentrations on both sides.

Osmotic pressure

pressure which needs to be
applied to a solution to prevent the inward flow of water across a semipermeable membrane

Hypotonic environment

Greater concentration of solute inside the cell

Hypertonic environment

Greater concentration of solute outside of the cell

Isotonic

Equal concentrations of solute inside and outside the cell

Higher salt concentration leads to

plasmolysis and cell death

plasmolysis

shrinking of protoplasm - its plasma membrane detaches from the cell wall

Halophiles 

Bacteria requires high salt concentration for growth 

Halotolerant

does not require a saline enviroment but can survive in one

Consequences of high PH

Breaks hydrogen bonds holding DNA together

Consequences of low PH

breaks the weak hydrogen bonds of protein side chains, changing the shape of the protein because increased hydrogen ions

Acidophile

pH 0-5.5

Neutrophile 

grows best around 7pH

Alkaliphile

grows best pH 8-11.5

Antibiotic susceptibility test

determines which antibiotics
or other antimicrobial agents are effective against a specific microorganism that is causing an infection.

Kirby-Bauer method

Antibiotic diffuses from a filter paper into agar plate. If antibiotic is effective, it will inhibit bacterial growth surrounding the disk creating zone of inhibition (ZOI)

Diameter of ZOI (in mm)

determines if the organism is Resistant (R), Intermediate (I), or Susceptible (S)

When do we use Mueller-Hinton Agar and why?

Mueller-Hinton Agar :
• General growth media that supports growth of most pathogens
• Highly reproducible results
• Low in thymidine and thymine, which inhibit some antibiotics

Antiseptics

Chemicals used on live tissues to control microbial growth Ex: isopropyl alcohol, mouthwash

Disinfectants

Chemical agents that are used on inanimate objects Ex: Bleach

Fomites

objects or materials which are likely to carry infection, such as clothes, utensils, and furniture.

Cytolysis 

The process where a cell swells and bursts due to an excessive influx of water.

Psychrophiles

<15 °C

Mesophiles

15 °C - 45 °C

Thermophiles

> 45 °C

hyperthermophiles

>80°C

Moist Heat

More effective than dry heat because it can penetrate microbial cells and denature proteins and melt lipids in cytoplasmic membrane

Moist heat example

Autoclave

Dry Heat

usually used to sterilize instruments.

Dry heat examples

Hot air sterilization

Thermal Death Time (Temp & time)

hortest exposure time to heat resulting in no growth after incubation at 37°C/48hr.

Effects of UV exposure

induces pyrimidine (thymine) dimers (Kink in backbone inhibits DNA polymerase activity
▪ Incorrect bases inserted to the DNA strand)

UV light is used to

steralize surfaces (short UV light) - Photoreactivation repair.

Horizontal gene transfer (name three ways)

The movement of genetic information
between organisms, a process that
includes the spread of antibiotic resistance
genes among bacteria (except for those
from parent to offspring), fueling pathogen
evolution                                                        Ex: Transformation, Conjuction, Transduction

Transformation

The process by which competent bacteria take up naked DNA from the environment.

Competent Cells

The ability of the cell to take up extracellular DNA from the environment

2 Types of Artificial Transformation

Chemical method: Calcium
chloride (CaCl2)
▪ Cells are repeatedly washed with
100 mM CaCl2 to remove all
traces of other salts (from the
growth media), then are heat-
shocked
▪ Calcium chloride is added to a
cell suspension, which promotes
the binding of plasmid DNA to
lipopolysaccharides (LPS) Physical method: Cells are repeatedly washed with a weak glycerol solution then treated with a brief electric shock, causing holes in the cell wall, through which DNA is drawn into the cell.

Plasmid ( or sex factors, conjugants, extra chromosomal replicons, or transfer factors )

Extra chromosomal DNA fragments in the cell They are double stranded structures.They can replicate independently

Structure of Plasmid (5)

Origin of replication (ori): a specific location in the strand where the replication process begins.
▪ Selective marker site: This region consists of
Antibiotic resistance genes which are useful in the identification and selection of bacteria that contain plasmids.
▪ Promoter region: Allows expression of the gene of interest
▪ Regulator: Controls expression of the gene of interest by acting on the promoter
▪ Multiple cloning sites: Insertion site for gene of interest

Gene Naming (nomenclature)

gene names are itilizised and start with lowercase letters (gfp, bia, ori), if multiple subunits - the subunit is at the end of the gene name is capalized (araC, lacZ)

Biofilms

multicellular communities held together by a self-produced extracellular matrix.

5 stages of biofilms

1.Reversible attachment: cells attach and detach from
the surface
2. Irreversible attachment: cell irreversibly attaches to
the surface via cell adhesion structures (like pili)
3. Maturation I: Slime production
4. Maturation II: the biofilm becomes encased in an
extracellular matrix
5. Release: cells within the biofilm are released.

Viable counting

A method to estimate the number of viable (alive and able to replicate) bacterial cells
in a cell suspension using serial dillution

CFU’s

Colony forming units - use CFU’s between 30-300 for counting

DF

Dilution factor - (Volume factor/total volume factor) x  (DF of previous tube)

FDF

Final dilution factor = volume plated (ml) x DF of counted plate

OCD

CFU of counted plate / FDF

Spontatnous mutations 

mistakes in DNA Replication, - Rate: 1 × 10^6 to 1 × 10^9

Induced Mutation

Mutations that are unnaturally forced through exposure to mutagens

Streptomycin gradient plate used for 

Used for isolation of antibiotic mutations

Streptomycin gradient plate is made with

Nutrient Agar with Blue dye on the bottom slant & Streptomycin agar as top slant

Defined Media

media composed of pure ingredients in carefully measured concentrations dissolved in double distilled water

Complex Media

Rich in nutrients that contain water soluble extracts of plant or animal tissue (e.g., enzymatically digested animal proteins such as peptone and tryptone)

why and what agar

A jelly-like substance consisting of polysaccharides obtained from the cell walls of some species of red algae -used to solidify media

What temperature is the liquid state of agar.

100°C

What temperature does agar solidifies?

44°C

Autoclaving requirements

121°C for 15 minutes at 15 psi

Macconkey’s Agar 

Type, what it’s used for, 

Selective & Differential - used to differentiate lactose fermenting GRAM NEGATIVE bacteria from lactose non-fermenting 

Selective: inhibit gram postive 

Differential: Turns red below pH 6.8 (fermenting bacteria turns lactose into lactic acid )and colorless higher than pH 6.8 (non-fermenting bacteria)

What type of media is this?

MacConkey Agar

MSA (Mannitol Salt Agar) - type and what is it used for 

Type: selective and Differential 

Used for isolation and indentification os Staphylococcus aureus 

Selective: High concentration of sodium chloride inhibits other bacterial organisms other than staph 

Differential: Mannitol fermentable carbohydrate - detectable by phenol red indicator 

Phenylethyl Alcohol Agar  (PEA) - Type and what is it used for?

Type: Selective 

Used to cultivate GRAM POSITIVE organisms 

Selective: PHENYLETHYL ALCOHOL is the active ingredient that inhibits the growth of Gram-negative orgnanisms (opp of MAC agar)

Eosin Methylene Blue Agar (EMB) - type and what is it used for?

Type: Selective, Differential, and Enrichment 

Selective: Eosin Y and methylene blue are Ph indicator dyes inhibit the growth of most Gram Postive organisms (only GRAM NEGATIVE grow)

Differential:

Lactose fermenting gram negative bacteria - purple or metallic green

Slow lactose-fermenters - pink 

Non-lactose fermenters - colorless or pink 

Enrichment: Sucrose and lactose utilized as fermentable carbohydrates which encourage growth of some gram negative bacteria 

Blood agar - type and what is it used for

Type: Enrichment and Differential

Used to culture fastidious (microorganisms that have complex or particular nutritional requirements and are difficult to grow in a laboratory) microorganisms and detect hemolysis

Differential: Different types of hemolysis

alpha- hemolysis: partial hemolysis, greenish opaque zone (beige)

beta-hemolysis: complete hemolysis, clear zone (ate up the RBC in agar) (bright yellow)

gamma- hemolysis: no hemolysis (dark red)

What type of agar is this?

Blood Agar - and tells us the bacteria is alpha hemolysis - which is partial hemolysis shown by the beige color

What are endospores and what is their purpose?

Dormant structure made in the cytoplasm and they are made to protect genetic information. They also have distinct endospore locations, central, sub terminal , and terminal. When conditions are better, they will return to their vegetative state

What are the arrows pointing to?

The red is a vegetative cell and the green circle is an endospore 

How do you stain an endospore?

Malachite green is used to stain the endospore and Safranin is used to stain the vegetative cell subsequently, only to GRAM POSITIVE

What are capsules?

Extracellular structures produced by bactera to allow them to adhere to surfaces, evade the immune system, protection, ect - HAVE TO DO CAPSULE STAINING (NEGATIVE STAINING)

What is negative staining?

Also known as capsule staining - use nigrosin that stains backround of slide and safranin to stain the underlying vegetative celll. - only GRAM NEGATIVE

Left to right of arrows

Nigrosin - dark purple, blue background

Vegetative cell - pink

Capsule - clear

Why use Acid fast staining? what is it?

Bacteria’s cell walls containing large amounts of waxy lipids called mycolic acid are highly resistant to normal staining techniques - You stain first with Carbol Fuchsin, which dyes the positive bacteria red, and then a counterstain with methylene blue, which shows the non-acid-fast bacteria as blue.

what stain ? what does this say about the bacteria?

Acid-fast stain 

A: Positive acid-fast stain bacteria- contain large amounts of waxy lipids - MYCOBACTERIUM 

B: Negative acid-fast stain bacteria - do not contain large amounts of mycolic acid

What is a colony?

A cluster of bacterial cells growing on solid media and is assumed grown from one bacterial cell

Colony morphology

Characteristics of a colony

Size

Shape

Color

Texture

Elevation

Margin

Difference between bacterial and fungal colonies 

Bacterial: usually small with defined margins (edges) 

Fungal: usually large with fuzzy or powdery appearance. Usually green-ish or white

Purpose of a streak plate?

To isolate bacterial colonies to obtain a PURE CULTURE bc usually bacteria are found in a mixed culture

Two types of stains are

Simple and Differential

What’s a simple stain?

Uses only one staining dye to see cells 

Helps identify size, shape, and arragement of bacterial cells 

what’s a differential stain ?

used two or more staining dyes to visualize cells with more than one color , can do everything of a simple + differentiate between cell types and structures

EX: Gram stain, capsule stain, endospore stain, acid-fast stain

Why and what is a Gram stain?

Differentiates bacteria based on peptidoglycan layer in cell wall,

Gram positive - thick cell wall and retain crystal violet and come out purple

Gram negative - thin cell wall and does not retain violet and counterstained pink

2 types of cell morphology (prokaryotic)

Coccus - round

Bacillus - rod shape

What stain and what does it say about the bacteria?

Gram stain with both gram positive cocci and gram negative Bacilli

What stain and what does it say about the bacteria?

Gram Stain - w/ gram postive bacilli

What stain and what does it say about the bacteria?

Gram Stain - w/ gram postive cocci

What stain and what does it say about the bacteria?

Gram stain w/ gram negative bacilli

What stain and what does it say about the bacteria?

Gram Stain w/ gram negative cocci

Name a gram postive bacteria

S. aureus, Streptococci, 

Name a gram negative bacterium

E.Coli, Salmonella

S. aureus or E. coli

E.coli - pink = gram neg and Bacilli shape

S. aureus or E. coli

S. aureus - purple = gram pos and Cocci shape

Who discovered the Gram Stain?

Hans Christian Gram

What is catalase?

an enzyme that reduces HYDROGEN PEROXIDE into water and oxygen 

Why can’t you use bacteria growing on blood agar for catalase test?

b/c contains RBC - form false positive

Why and what is a catalase test?

only GRAM POSITIVE - differentietes if produces enzyme catalase 

add hydrogen peroxide and see bubbles 

EX: Staph 

what is a Starch hydrolysis test, most common bacteria, and dye?

tests for extracellular enzyme,s also called exoenzymes - they help break down larger macromolecules

usually Bacillus

Gram’s iodine - reacts with starch to make a dark green/ blue color, and if an enzyme present will be clear

48 hour test

What is this plate? and what does it say about the bacteria?

Starch hydrolysis plate - does not produce exoenzymes because dark green = negative result 

What is this plate? and what does it say about the bacteria?

Starch hydrolysis plate - does produce extracellular enzymes bc light color = positive results

Why Citrate Utilization Test and what dye?

utilize citrate as a carbon source and inorganic ammonium salts as the sole source of nitrogen - if can grow will be blue and contain an enzyme citrate-permease and break down ammonia increases pH

BROMOTHYMOL BLUE pH indicator when pH is above 7.6

Green - negative = no citrate-permease, cant breakdown citrate and pH below 7.6

Blue - positive = Citrate-permease present, can breakdown citrate and pH above 7.6

24 HOUR TEST

What is a motility test

Differentiates between motile and non motile organisms - have flagella - semi solid then other media

Motile organisms reduce the colorless tetrazolium chloride (TC) to a red-colored product called formazan  24 HOUR TEST