1. Nitrogen
2. Phosphorus 
3. carbon
4. minerals
5. proper pH and osmotic conditions 

1. Spred auxotrophic S. typhimurium over agar
2. make control plates
3. place disk soaked with potential mutagen in the middle of the plate 
4. count the # of colonies 

Benzne gave a positive Ames Test result and caused a tumor in mice. Based on these two results, you could determine that benzen is:

Both a and b are correct

What dye will be used to quantify biofilm formation?

a. Methylene blue

b. Safranin

c. Crystal Violet

d. Any dye can be used

• Uvrb= cannot repair DNA mutation
• Rfa= cannot synthesize a complete cell wall
• His-= cannot synthesize histidine

Compare and contrast Biofilm and cellulose pellicle:

Compostion 

Biofilm:

Polysaccharide

Cellulose pellicle:

Cellulose

Compare and contrast Biofilm and cellulose pellicle:

Organism responisble for the bioflim/pellicle production

Bioflim:

S. aureus

Pellicle:

Acetobacter Acetti 

Compare and contrast Biofilm and cellulose pellicle:

Purpose or advantage of the biofilm/ pellicle production

Biofilm:

Due to close proximity increase frequency of lateral gene transfer thru conjugation thus they are more resistant to antibiotics. 

Pellicle:

Access to O₂

Protection from U.V. 

TRUE/ FALSE

An example of a "reversion/supressor/back mutation" is the mutation that reverts auxotrophic mutants back to prototrophic state. 

1. Attachment (adsorption): virus become attach to host cell
2. Penetration: virus injects its nucleic acid into the host cell
3. Synthesis: take over host machinery and begins to replicate viral DNA 
4. Assembly: assembly and packaging of mature viral particles 
5. Lysis: release viral particles (the cell is killed in order to exit)

You are investigating the mechanisms of infection by bacteriophages and have obtained a suspension of the T4 bacteriophage of unknown concentration for use in your study. You perform a plaque count assay with 1:100 dilution of your phage suspension by plating 0.1 ml of phage and obtain the results below. Determine the original concentraion of phage in your suspension. Show all your work for full credit.

(There are 13 PFU's)

                             13 PFU's    x 100= 1.3 x 10⁴ PFU/ml

                                0.1 ml

                                 # PFU's x D.F.= [orginal]

Volume plated

1. Penicillin [ in lab]
2. Ampicillin [in lab] 
3. Cephalosporin 

True/False

An example of a "Reversion mutation" is the mutation that reverts auxotrophic mutants (eg. his-mutants) back to wild type by restoring the original DNA sequence. 

True/ False

A supressor mutation rescues the phenotpe or a prevous mutation and results in a WT genotype 

True/ False

A PFU, or Plaque forming unit, is a colony of bacteria infected with a bacteriophage. 

The ames test involves spreading a mutated form of a bacterium in soft agar over a layer of minimal media. We then put a disk containing our chemical on the plate. We allow the plate to incubate and then observe the plate for growth and notice 6 small colonies aroudn the filter disk. Your positive control has numerous colonies on its plate and the negative control only has 2 CFU's on the plate. Answer the following: 

1. Why is there growth on the negative control plate? Does the growth on your negative control plate mean that your negative control is a mutagen, explain?

There is growth because some bacteria were able to revert back to prototrophy, due to spontaneous mutation. This allowed them to synthesis the 3 mutations that they would not normally synthesize. 

The ames test involves spreading a mutated form of a bacterium in soft agar over a layer of minimal media. We then put a disk containing our chemical on the plate. We allow the plate to incubate and then observe the plate for growth and notice 6 small colonies aroudn the filter disk. Your positive control has numerous colonies on its plate and the negative control only has 2 CFU's on the plate. Answer the following: 

2. What do these results tell you about your test substance, how did you come up with this conclusion?

True/ False

The rfa mutation in the bacteria used in the Amest test prevents the cells from synthesizing a complete cell wall.

True / False

The bacteria carrying the mutations necessary for the Ames test is Salmonella sanguis. 

False

S. typhimurium 

What are the disadvantages of the kirby bauer test?

• Basic dyes
• Acid dyes

True/ False

A working culture is a culture that is store at a temperature that prevents microbial growth

True / False

Klebisella has two membranes and a thin peptidoglycan layer, thus it is a gram + bacteria 

True/ False

Aseptic techniques are important for preventing contamination of pure bacterial cultures

• Methylene blue- staind bacteria
• Nigrosine- stains background 

1. Denaturing: ~95 °C: H-bonds break 
2. Anneling: ~64 °C:allow annealing of primers
3. Elongation: ~74 °C: TAQ polymerase binds and extends DNA 

• Catalase
• O₂
• H₂O 

• Exoenzyme
• Starch

• Differential staining: uses safranin, crystal violet, iodine,a nd alcohol to dye cells. Depending on cell composition, the dyes will stain the sample a different color. Gram +: purple, and gram -: pink. Size and shape can also be determined. 
• Capsule staining: [ uses a simple ( stains bacteria) and neg. stain(stains background) ] uses methylene blue and negrosine black dyes. The background is negative stain this shows the overall morphoplogy of the bacteria. This is used on delicate cells,such as spirochettes and capsule. 

250 x 10= 25000 cells/ml

0.1 ml

35 x 100= 35000 cells/ml

0.1 ml

3 x 1000= 30000 cells/ml

0.1 ml

25000+35000+30000=30000 cells/ml  

             3

You want to investigate the effect of higher pH on bacterial gene expression. You have obtained a pre-labels microarray chip containing complementary sequences to each of the genes in the bacterial cell that you are using. You have designed your experiment as follows:

You have two cell cultures: 

A has cells at pH 7 (optimum temperature) and B has cells at pH 9. You allow the cultures to incubate long enough to observe different gene expression. You then extract the mRNA from the cells in both cultures and prepare cDNA ( DNA strands that are complementary to the mRNA strands that were present in the cell). These cDNA strands are fluroescently labeled; cDNA from cells in culture A are red while cDNA from cells in culture B are green. You allow the cDNA to hybridize with the sequences on the microarray chip and look at the chip with a fluorescent microscope. 

a. What does a red spot indicate on the plate?

b. What does the green spot indicate on the plate?

c. What does the yellow spot indicate on the plate? 

The biofilm formation requires four steps:

In step one ____ bacteria approach a solid surface taht is accessible to bacteria and maintains the minimm amount of nutrients needed for survival. 

In step two bacteria become loosely associated with the surface forming an attached cell. 

In step three cells form aggregates and a stable attachment to the surface forming a ______. 

In step four, a _____ matrix is excreted by the bacteria encasing the biofilm. 

Its role is to detach the biofilm from its surface so that it gets suspended in the liquid so that you can take the od reading. 

Will you be able to obtain data point(s) corresponding to the death phase of the bacterial growth curve by using the techniques performed in the lab (measuring the optical density or absorbance at 30 min time intervals) explain?

Death phase cannot be measured with turbidimetry, because turbidimetry cannot distinguish between dead or alive cells. 

gen. time=  time  =          T_f - T_i =    

                 # of gen       3.3 log  n_f

                                               n_i

                                  10000000-100000 = 4.3 x 10 ⁶

                                         3.3 log 5 

Bacteria that can respond by focusing their direction of travel toward or away from a chemical stimulus exhibit:

a. Brownian Motion

b. Chemotaxis

c. Motility

d. Have many cilia 

Please explain how the STYO9 green fluroescent dye and the propidum idoide work together to stain a bacterial culture live and dead. Make sure to also include which dye lables live and dead cells the resulting color. 

First you use the STYO9 green fluorescent to stain all of the cells. Then you use the propidum to stain all of the cells that have broken membranes. The propidum will stain the dead cells red. Therefore, the green ones are alive. 

# of CFUs  = [ conc. of last dilution] 

Vol. plated

# of CFUs =2.4 x 10 ³ org/ml 

    0.2 ml

480 CFUs/ ml 

aggregates of bacteria attached to a surface encasd in a structured (3-D) polysaccharide matrix 

Where do biofilms form?

any surface that is accessible to bacteria and maintains the minimum nutrients needed for survival

• rocks in streams
• teeth (plaque)
• catheter/IV in a patient's arm 
• water pipes

1. Planktonic (free swimming) 
2. Bacteria become attach to surface
3. micro-colony is formed
4. polysaccharide matris is secreted, encasing the bacteria 

True/ False

A viral infection can be treatd with an antibiotic 

• more readily acquire transmissible, genetic elements (increase frequency of conjugation= increased rates of evolution) 

Lysogenic: integrate gene 

- last longer 

Lytic: host cell is lysis and releases new phage particles and repeats 

Supressor:a second mutation that restores the function lost by a primary function. 

Revergent: a mutation that precisely restores a mutant DNA sequence to a WT DNA sequence

 Stock:

True/ false

Is 95% ethanol used to kill?

True/ False

Virus are non cellular 

True/ False

Microarray chip- single genes 

α- hemolysis: partial digestion

β- hemolysis: complete digestion

Υ- hemolysis: no digestion

• unstable 
• no OH group

Primary: articles [ the original source] 

Secondary: books, journal reviews [not the original source]

Media composed of designated amounts of specific compounds and exact contents are known. 

Ex. minimal media: provides only nutrients necessary for growth of the microbe

Contains nutrient rich substances such as yeast extract, peptone, tryptone exact constituent are unknown . 

Ex. TSA

Allows growth of one group of bacteria while inhibiting some other group 

Ex. MSA

A certain chemical in the medium cause change in the medium after the bacteria growth

Ex. EMB differentiates between lactose and non-lactose.

Supports the growth of fastidous bacteria by providing specific nutrients 

Ex. Blood agar

• H2O agar
• TSA
• CSM

contains yeast extract, peptone, mannitol

Acetobacter→ Acetti→ produce ~2um of cellulose strand/min

1. Cellulose traps aerobic bacteria and provide access to oxygen

2. Protection from UV→ less mutations and increse in survival 

SEM