BIOC17 - Lab Content

Lab Manual + Lab Review Slides

What is a simple microscope?

A microscope that uses one lens to magnify to about 10x-30x

What is a compound microscope?

A microscope that uses two or more lenses to magnify >1000x

What is the difference between a monocular, binocular, and trinocular microscope?

A monocular microscope has a singular eyepiece lens, a binocular has two eyepiece lenses, and a trinocular has two eyepiece lenses and a third lens to mount a camera on

What is the difference between dark field and bright field light microscopy?

In dark field microscopy the specimen is lit while the background remains dark, and in bright field microscopy the field is bright while the specimen is dark

What is the difference between magnification and resolution?

Magnification is how enlarged a specimen appears while resolution is how clearly and distinctly fine details can be seen

What are basic stains?

Stains that are positively charged

What are some examples of basic stains?

Crystal violet, safranin, and methylene blue

What are acidic stains?

Stains that are negatively charged

What are some examples of acidic stains?

Nigrosine, eosin, Congo red, and India ink

Why are basic stains more commonly used?

Because the cell membrane is negatively charged, basic dyes bind to the cell surface and stain them while acidic dyes are repelled

In what cases is heat fixation avoided?

When performing negative stains, capsule stains, and when alive cells are wanted (hanging drop)

What is the purpose of heat fixation?

Kill the cells, coagulate the cytoplasmic proteins to increase visibility, and help cells adhere to the slide

What are the consequences of heat fixation?

It will distort and dehydrate/shrink cells

What is a mordant?

A chemical agent that helps bind dyes more tightly to cellular structures

What is the role of the Ryu stain in flagella staining?

Ryu stain acts as a mordant

What is the difference between carbolfuchsin stain used in Ziehl-Neelsen and Kinyoun’s cold procedures for Acid-Fast staining?

Due to the lack of heat as a mordant in Kinyoun’s procedure the carbolfuchsin has a higher phenol concentration

Can a capsule layer on bacteria be dissolved by alcohol in Gram staining?

No, capsules are made of carbohydrate layers and alcohol does not dissolve those

Why do we not heat-fix in capsule staining techniques?

Heat fixation can cause the cytoplasm to shrink away from the cell wall creating a false positive for the test

Which has higher agar concentration between LB agar and motility test agar?

LB agar has a much higher agar concentration

What is the role of tetrazolium salt in motility agar?

Tetrazolium salt is used by the bacteria as an electron acceptor as they grow. When it’s reduced it becomes red and precipitates out → acts as an indicator of motile bacteria

Where do we start streaking on an agar slant surface?

Start streaking at the bottom of the slant

Why cover bacterial smears with a piece of blotting paper in endospore staining?

To prevent the stain from evaporating during heating

What is the consequence of overstaining with crystal violet in Gram staining?

The results will give an overestimate of Gram (+) cells (all purple) as the alcohol won’t be able to decolorize well

What is the consequence of over-incubation with decolorizer during Gram staining?

The results will overestimate how many Gram (-) cells there are/give a false negative → cells are red

What is the consequence of skipping the washing step in endospore staining?

The counter stain won’t work

What is the role of NYZ agar in promoting viral infection for plaque assay?

NYZ agar contains mannose and MgSO₄ → mannose-phosphate receptors are needed for viral uptake and MgSO₄ helps interaction between the mannose receptors and ligand (virus)

Are the phages we used temperate or non-temperate?

Lambda-pages are temperate phages

Why should phage: bacteria ratio be kept very low in plaque assays?

This ensures that each resulting plaque originates from a single viral particle and prevents plaques from merging with each other

Which E. coli strain did we use for the bacterial transformation experiment and why?

HB101, this strain is not resistant to ampicillin and lacks endogenous restriction enzymes making it ideal for transformation experiments

What factors determine transformation effciency?

The size of the plasmid, cell competence, and purity of the plasmid DNA

What is the arabinose operon responsible for?

The genes are responsible for the catabolism of arabinose

What is the role of araC in the operon?

araC is the regulator for the operon, when arabinose is present it induces the operon to produce the BAD genes

Is the arabinose operon inducible or repressible?

It is an inducible operon

Does pGLO have araBAD genes? Why or why not?

No the araBAD genes are replaced with the GFP gene under the P_BAD promoter because if the araBAD genes were there catabolism of arabinose would occur and then the operon would naturally be turned off

What is the consequence of agitation during conjugation?

Some agitation can increase the number of collisions between bacteria allowed for increased levels of conjugation, however excessive agitation can lead to high forces that pull pairs apart

Why was E. coli strain J-53R used as the recipient strain?

Due to it’s auxotrophic nature it ensures that the doubly-resistant cells can’t grow in nature, which stops the spread of further resistance

Why did we use Tryptic soy broth to grow J-53R?

TSB is high is amino acids which the strain requires to grow

What factors affect generation time?

Nutrient availability, pH levels, oxygen levels, temperature, are some key factors

Why is generation time measured in the log-phase?

Because this is the period when bacteria are constantly dividing at their maximum rate, when plotted on log paper it gives a straight line

Why is the term turbidometry used instead of spectrophotometry or colorimetry?

Turbidity is used as we are measuring the amount of light the cells scatter rather than light that is absorbed and since cells are uncoloured the term colorimetry doesn’t apply either

What does a typical OD₆₀₀ value of 1.0 correspond to in cells/mL for E.coli?

It’s about 8×10⁸ - 1×10⁹ cells/mL

What are some direct count methods?

Counting chambers, flow cytometry, and electronic counters

What are some indirect count methods?

Turbidometry, viable cell count, most probable number test, and microbial dry weight

What wavelength is used for turbidometry for bacterial cultures?

600nm

What is the range of detection for direct count methods?

<10⁷ cell/mL

What is the range of detection for indirect count methods?

≥10⁷ cell/mL

How many large and small squares are there in a Petroff-Hauser counting chamber?

20 large squares and 16 small squares

How much volume does a small square in a Petroff-Hauser hold?

5x10^-8 mL

What is the equation for calculating original cell density for a Petroff-Hauser counting chamber?

OCD = # of cell counted/(# of squares)*(dilution factor)*(volume of a single small square)

What is the optimal density per small square in a Petroff-Hauser?

5-15 cells

What’s different about a Petroff-Hauser counting chamber coverslip?

It’s thicker and a specific depth to allow for clarity when counting

Why use Mueller-Hinton agar for Kirby-Bauer’s disc diffusion test?

• Allows growth of all organisms including fastidious microbes

• No chemicals to inhibit antibiotic action

• Contains starch to absorb toxins released from bacteria → minimizes interference with antibiotics

• Allows better diffusion of antibiotics

How does penicillin affect gram-positive bacteria vs gram-negative bacteria?

Penicillin works on gram-positive but not on gram-negative

How do you determine if an antibiotic is bacteriostatic or bactericidal?

Swabbing the inhibition zones from a Kirby-Bauer diffusion test and inoculating fresh agar to allow growth would show if the antibiotic is bacteriostatic or bactericidal

What is meant by McFarland Standard?

It’s a fine precipitate formed by barium chloride and sulfuric acid, it’s used to adjust the turbidity of the bacterial suspension in tubes before a Kirby-Bauer disc diffusion test

Why is a McFarland Standard used?

It standardizes the approximate number of bacteria which means that measuring the zone of inhibition is meaningful.

What is the purpose behind the cefinase test?

If the organism swabbed onto the disc produces β-lactamase it will breakdown the cephalosporin nitrocefin and produce a pink colour

Is a cefinase positive bacteria suitable to use for transformation with pGLO?

No, as the positive result means that it will breakdown β-lactam antibiotics (like ampicillin) inherently and this makes the ampicillin plates ineffective at selecting for transformants

What is the general difference between disinfectants and antiseptics?

Disinfectants are much stronger and will kill all organisms and are used on surfaces/objects, they aren’t suitable to be used on/in living systems as they’ll kill non-pathogens as well

Which showed higher catalase activity S. epidermidis or E.coli?

S. epidermidis has higher activity

What is the effect of H₂O₂ on aerobic vs anaerobic bacteria?

Aerobic bacteria have catalase which is able to breakdown hydrogen peroxide into water and oxygen gas and so it’s not effective on aerobic bacteria but anerobic bacteria lack this enzyme, making H₂O₂ very effective

Why could satellite colonies be observed surrounding antibiotic-resistant bacterial colonies with over-incubation?

With over-incubation the resistant bacteria begin to breakdown the antibiotic in the area surrounding the colony allowing for small non-antibiotic-resistant satellite colonies to grow

How does UV exposure effect bacterial physiology?

UV exposure leads to DNA damage by thymine-thymine dimers which distort the structure and stop replication and transcription → leads to cell death

What makes endospores resistant to UV induced DNA damage?

In endospores small acid-soluble proteins tightly coat the DNA to physically protect it, this changes the shape of the DNA so that thymine dimers aren’t produced

Why do we rest the plate lids on alcohol-soaked paper towels?

It prevents contamination

What’s a skin microbe that we’ve been using in labs?

S. epidermidis

What hand-washing conditions were the most effective?

Proper handwashing, drying hands completely after, and using hot water

What goes wrong at very low temperatures for mesophiles?

Plasma membranes can solidify and enzymes stop working

What goes wrong at very high temperatures for mesophiles?

Membranes, enzymes and DNA start to denature

What kind of adaptations do thermophilic bacteria have in their plasma membrane, cell structure and composition?

Archaea have ether linkages in their membranes which are more heat resistant, a less fluid membrane due to the isoprene units, and have S-layers, polysaccharide layers, and/or protein sheaths that protect from heat.

What goes wrong at very low or high pH for neutrophiles?

Extreme pH levels affect H-bonding in macromolecules, modify amino-acid functional groups, and affect protein folding which promotes denaturation

What is an example of a non-osmotolerant bacteria?

E. coli

What is an example of a common osmotolerant bacterial species?

Staphylococcus spp.

What’s the difference between thermal death time and decimal reduction value?

TDT is the time needed to kill a specific number of microbes at a specific temperature while D-value is the time needed to reduce the number of viable cells in a population by 90% at a specific temperature

How does Mannitol salt agar (MSA) allow growth of gram-positive cocci over gram-negative bacteria?

The salt content selects for osmotolerant Staphylococci. The differential component is the mannitol, microbes capable of mannitol fermentation will drop the pH making the phenol red indicator turn yellow

Which components of MacConkey agar are selective and which are differential and how?

The bile salts and crystal violet select for gram-negative bacteria while the lactose differentiates lactose-fermenters from non-fermenters. With fermentation the pH drops and a red colour appears.

What are coliform bacteria?

A subgroup of Enterobacteriaceae that produce gas from lactose fermentation

What two steps are involved in the hydrolysis of gelatin?

Gelatinase breaks gelatin down into polypeptide chains and then further into amino acids

What component of the Snyder Test indicates the presence of dental decay causing bacteria? How?

The pH indicator, bromocresol green, turns yellow in the case of acid which is an indication of sugar fermentation which is done by dental decay causing bacteria

What components of DNase agar help detect the secretion of DNase in extracellular media?

It contains DNA bound to methyl green, when the DNA is hydrolyzed the methyl green is released. At the neutral pH the indicator turns colourless resulting in a clear halo around the bacteria

Which of these bacteria are DNase positive - S. epidermidis, S. aureus, Serratia marcescens?

S. aureus and Serratia marcescens are DNase positive

What is blood agar made of?

Tryptic soy medium and sheep’s blood

What kind of exotoxins are produced by gram-positive hemolytic bacteria?

Streptolysin O (oxygen-labile) and Streptolysin S (oxygen stable) but are both optimally expressed under anaerobic conditions

What kind of challenges do biofilms present in disinfection of the environment?

Biofilms are infamous for forming on invasive medical equipment (catheters) and causing hospital infections