Microbiology Lab Exam 2

What is ph?

The negative of the hydrogen ion concentration in a solution.

-Log[H+]

Acidic

Ph below 7

Alkaline

PH above 7

Neutral Ph

PH of 7

Acididophiles

Below pH 5.5

Alkaphiles

PH above 8.5

Neutrophiles

Between 5.5 and 8.5

What happens,If you put a microbe in a ph that is too high or too low?

disrupt the membrane and damage enzymes

Isotonic solution

no water is going to be moving back and forth, if it does there’s no net change 

Hypotonic

waters moves in

Hypertonic

water moves out

No growth

clear

Poor growth

can see through it

Good growth

can read print through it

Excellent growth

cannot read print through it

Hyperthermophiles

Above 80 degrees

Psychotroph

0-37 degrees; Can grow outside of its temperature, can grow in the refrigerator and it can cause food spoilage

Obligate aerobes

Require oxygen for growth because they carry out respiratory metabolism in which the final electron acceptor is oxygen

Microaerophiles

Prefer to grow in oxygen concentrations of 5-10%.

Facultative anaerobes

Can grow with or without oxygen but will use oxygen if present.

Aerotolerant Aerobe

Grow whether oxygen is present or not, but they usually have a strictly fermentative metabolism

Obligate Anaerobe

Can not tolerate oxygen, its toxic to them so they will not grow in the presence of oxygen

How do we culture microbes

By using FTM (Fluid thioglycollate) medium

What are the 4 enzymes that detoxify harmful oxygen enzymes

Superoxide, Dismutase, Catalase and Peroxidase

What does FTM do?

binds oxygen and oxygen is given off when the tubes are heated

Kirby-Bauer test (diffusion test)

Procedure that involves spreading test bacterium on the surface of Muller Hinton agar

Disks are infused with a set amount of specific drug placed on the agar surface

Plates are incubated

collected data is often presented in an antibiogram

Zone of inhibition

a clear area around the disk where no microbe is growing, Diameter is measured.

Susceptible

zone of inhibition falls in the susceptible range

Resistant

zone of inhibition falls in the resistant range

Intermediate

borderline response (only use at the last resort)

Highly Standardized

• pH 7.2-7.4

• 4mm thick

• Mueller-Hinton Agar (MHA)

• Antibiotic concentration

• Bacterial Concentration

Antibiotics work in different ways:

Targets cell wall, cell membrane, cellular synthetic processes and proteins.

Ciprofloxacin

Inhibits DNA Gyrase

Polymyxin

Damage cell membranes

Penicillin

Inhibits transpeptidase

Trimethoprim

Inhibits folic acid

Protein Synthesis Inhibitor

Streptomycin:Binds 30S ribosome, causes misreading of mRNA

Tetracycline: Binds 30S ribosome: inhibits tRNA from binding A-site

Chloramphenicol: Binds 50S ribosome; inhibits peptide bond formation

UV light damages DNA by causing ____________ dimers.

Pyrimidine

DNA absorbs UV light at _______.

260nm

2 types of DNA repair:

Light repair: Uses photolyase to break the pyrimidine dimer bonds

Dark Repair: Endonuclease nicks the DNA near the DNA damage

Helicase removes the damaged fragment

DNA polymerase copies new DNA

DNA ligase seals DNA fragments together

pGlo experiment

CaCl2:Weakens cell wall and neutralizes the negative charge of bacteria

HB101 E.coli are used beause they accept pGlo and express pGlo plasmid

Heat shock is done to open pores in the E.coli to let pGlo in.

Expected results

LB plate with no pGlo(-DNA): No Growth. This is your viability control. If there’s no growth the cells are dead.

LB/AMP plate no pGlo (+DNA): Growth, the cells are resistant to Ampicillin

LB/AMP/ARA plate with pGlo (+DNA): Growth and green fluorescence. The bacteria are resistant to to Ampicillin and the ARA (arabinose the Green Fluorescent Protein to be expressed.

Components of PGlo plasmid

Bla gene: Codes for Beta-lactamase, This makes bacteria resistant to Ampicillin

pBAD: This is a promoter that binds RNA polymerase

AraC: THis will bind to arabinose

GFP gene: This gene codes for the Green Flurorescent Proten

In covered area there should be

continuous growth; no DNA damage occurs

Clearing with no growth

DNA damage and NO repair

Environmental factors effecting microbial growth

PH, Osmotic, Temperature, Aerotolerance

If you place a microbe at a Temperature higher than its range

The enzymes and proteins denature

If you place a microbe in temperature lower than its range

Metabolism slows down

Streptomycin

Binds 30s ribosomes; causes mRNA to be read incorrectly

Tetracycline

Binds 30s ribosome; inhibits tRNA from binding A-site

Chloramphenicol

Binds 50s ribosome; inhibits peptide bond formation

In the covered area there should be

continuous growth; no DNA damage occurs

In the exposed area

clearing with single colonies: this indicates there is DNA damage and repair

Clearing with NO growth: DNA damage and NO repair

Clearing with single colonies

This indicates there is DNA damage and repair