Microbiology Laboratory Techniques and Applications
Review again:
Chapter one:
There are three main hazards in the microbiology lab, they include,
Biological hazards
Physical hazards
Chemical hazards
Biological hazards are high concnetration of bacteria
Chemical hazards are ethanol, alcohol, and testing reagents
Physical hazards can cause damage either through direct or indirect contact.
Chapter five:
IF you want to grow bacteria in a lab you must provide all of their suitable growth conditions.
The medium in which all of the suitable growth factors are provided in are called a growth medium.
A lot of mediums are solidified using a agar
Agar is a colloidal polysaccharide, or a galactose polymer obtained from the cell wall of red algae
Agar provides no nutritional value but is simply used to solidify the medium.
Agar is used within these mixtures that are used in growth mediums because of its unique properties….
It does not completely melt until it reaches 100 degrees celsius and it does not soldifify until its cooled to 45 degrees celsius.
LB is made out of Yeast extract, tryptone, and NaCl
Yeast extract: basically everything in a yeast cell: proteins, nutrients, amino acids, vitamins, minerals except without the cell wall
Tryptone: A pancreatic digest of the casein protein
NaCl: Used to maintain a certain ionic charge in the broth.
Chapter 6:
Proper aseptic technique will decrease the risk of contamination.
There are two main methods to asceptically transfer cultures to media.
Loop/needle transfer:
Solid to solid
Solid to liquid
Liquid to solid
Liquid to liquid
Pipette transfer
Liquid to solid
Liquid to liquid
What is a liquid to liquid transfer:
From liquid culture to liquid medium
E.G: pond to luria broth
If you are transferring from a liquid to a slant or an agar plate you will use a loop transfer
Liquid to solid needle transfer:
A tool like a loop is utilized however it does not have a loop at the end.
Stick it into the middle of the sterile media up until ¼ away from the bottom.
Chapter 12:
Gram stain:
The most common differential stain is the gram stain….
The gram stain allows us to distinguish bacteria based on size, shape, and arrangement.
Instead of shape say morphology…
But can also distinguish between gram-positive and gram-negative bacteria.
Gram positive and gram negative bacteria stain differently because of fundamental differences in the structure of their cell wall.
The gram positive cell wall consists of multiple layers of peptidoglycan
A gram positive cell wall will be 60-90% peptidoglycan while a gram negative cell wall will be 10-20% peptidedoglycan
A gram negative cell wall will have less peptidoglycan but also have a lps layer (lipopolysachharide) layer.
Gram staining is composed of four steps:
Primary stain (crystal violet)
Crystal violet is a basic stain
A basic stain has a positively charged chromophore so its a basic stain.
Iodine (mordant)
The mordant is important for the formation of the CV-Iodine complex which is hard to wash off.
95% ethanol
The ethanol serves as the differential step. Gram negative bacteria will not retain the violet color, while gram positive bacteria will.
Safranin
Counter stain
Only gram positive keep the safranin color as they lost the CV stain.
The theory behind why gram positive bacteria keep the CV after being exposed to the 95% iodine solution is because the CV-I complex goes deep within the cell wall and cannot easily be washed away by the ethanol.
In gram positive bacteria the LPS layer is washed away by the ethanol.
Chapter 11:
Chapter 5:
Agar:
So agar is usually used to solidify a medium, its a colloidal polysaccharide, specifically a galactose polymer which is obtained from the cell wall of red algae.
Agar has no nutritional value, but is utilized to solidify a medium.
Agar is used because of its physical properties it doesnt completely melt till 100 degrees celsius and it remains liquid up until 45 degrees celsius.
If medium has agar you have to boil it to mix the ingredients.
Before the medium can be poured it muset first be sterilized. It can be sterilized through autoclaving which occurs at 121 degrees celsius @ 15 psi.
LB is made up of yeast extract, tryptone, and NaCl.
NaCl keeps the broth at a certain ionic strength.
Chapter 6:
Proper aseptic technique will help reduce the risk of contamination.
Loop transfer
Solid to liquid
Liquid to solid
Liquid to liquid
Solid to solid
Pipette transfer:
Liquid to liquid
Liquid to solid
Chapter 7:
There are two environmental factors which are heavily important for bacterial growth:
Physical
Nutritional
Physical factors include:
pH
Temperature
osmotic pressure
Gaseous requirements
Each bacterial species has its own optimum growth temperature.
psychorphiles : -5 degrees celsius to 20
Mesophiles 20-45
Thermophiles 45+
Cultures obtained from the environment are incubated at a temperature of 25 while cultures obtained from internal body sources are incubated at 37 degrees celsius
Nutritional requirements include water, sources of carbon and nitrogen, minerals and organic growth factors.
In the laboratory cultures are grown on mediums
There are liquid, solid, and semi sold cultures
Solid has 1.5% agar and semi solid has .7% agar
Liquid media is used to propagate large numbers of bacteria, whereas solid mediums are used for the observation of colkony apperareance, pure culture isolation, and short time storage of c ulturesm,
There are two types of mediums…
A complex/rich medium: has everything the bacteria needs to grow
A minimal/defined medium: Only has essential nutrients.
Bacterial growth:
Bacteria grow through binary fission
E. Coli divides every 20 mins
Growth stages
Lag
Log
Stationary
Death
In a broth medium u can determine the number of cells by looking at the OD of the medium
Od and density is directly proportional.
The spectrophotometer tranasmits a beam of light at a single wavelength through a liquid culturter and measures the amount of light that is scattered by the cells in the medium
Od for e.Coli is 1*10^8
Chapter 8:
Simple microscopes are just magnifying glasses.
It uses a simple biconvex magnifying glass to magnify an image.
When two magnifying glasses are used together the magnification ismultiplied and its called a compound microscope.
Resolving power limits a microscope.
Resolving power is defined as the ability to distinguish objects in a zoomed in image. You can differentiate them
d= resolving power
d= wavelength/2*na
As the value for D continues to decrease the better or more clear images will be
Immersion oil is meant to fill in the space between the lens and the object being looked at.
The oil and the lens have the same refractive index.
The microscope used in lab are binoculars, compound, and bright field.
Bright field means the object appears dark in a light background.
The objective lens are designed to be parfocal, which means that focus does not change as you switch objective lenses.
Chapter 13:
Carbohydrate Fermentation
All living organisms require organic and inorganic compounds for growth and reproduction.
Microorganisms obtain these compounds from the surrounding environment and transport them into the cell, where they are either used for:
Synthesis of building blocks
For energy
Carbohydrates are the most common source of energy for most chemoheterotrophic microorganisms.
Microorganisms utilize carbohydrates in either one of the following two pathways:
Fermentation
Respiration
Respiration can be broken into three steps:
Glycolysis
Krebs cycle
Oxidative phosphorylation
Fermentation does not require oxygen.
Fermentation is a redux reaction.
Oxidation occurs first
Then reduction.
Fermentation can be either aerobic or anaerobic.
Aerobic respiration is when oxygen is used as the oxygen acceptor.
Anaerobic respiration is when an other inorganic molecule is used as the electron acceptor including nitrate and sulfate.
During fermentation the glucose or sugar loses its electrons to the electron acceptor, this is the oxidation step. —> results in the formation of pyruvate molecules which are then reduced into an alcohol or acid. ( reduction process may result in the release of a gas.
Remember most carbohydrate fermentation include the breaking of disaccharide sugars into monosaccharides.
E.G: lactose —-> glucose and galactose
Phenol Red Broth: Is an all purpose differential medium which will appear yellow in acidic conditions (below 6.8), red at neutral ph (7), and pink at ph’s above 7.4
Peptones provide nutrients for all bacteria and include pH indicators which change the color of the medium depending on what the bacteria breaks down.
If peptide is broken down, ammonia is produced, which increases pH.
Alcaligenes Faecalies will always turn the medium alkaline, does not ferment.
Alca=alkaline
Remember that it’s extremely important for the tubes to be incubated no more than 48 hours, because once the carbohydrate is gone the peptone will be utilized.
Common sugars used in the PRB:
Lactose
Glucose & Galactose
Sucrose
Fructose and glucose
Glucose
Chapter 14:
A lot of gut bacteria are gram negative anaerobes of the Baacteriodes, followed by the faculat
Chapter 15:
Litmus milk= medium
The litmus milk test tests the ability of bacteria to transform the milk components into various different metabolic end products.
The litmus milk is composed of two main ingredients casein and lactose.
Four basic reactions can occur in a litmus tube.
Lactose fermentation
Lactose fermentation is a redox reaction.
Defining characteristics of reduction and oxidation reactions. The reaction may possess one or more of the following.
Reduction | Oxidation |
-O | +O |
+electrons | -electrons |
+H | -H |
Lactose fermentation can only occur in bacteria that possess the lac operon.
Bacteria that possess the lac operon have the lac Z gene which codes for B-galactosidase which breaks down lactose into glucose and galactose.
Glucose is then oxidized in the Embden Meyerhoff pathway to form pyruvate.
The pyruvate is then reduced into lactic acid.
The lactic acid is what causes the pH change in the medium which results in the pink color change.
Sometimes during the fermentation of the lactose a gas such as O2 or CO2 is produced. You know a gas formed if the acid curd has fissures in it.
The fermentation of lactose first involves an oxidation reaction and then a reduction.
If too much lactic acid is produced then the casein in the milk will become an acid curd.
Litmus Reduction:
Usually associated with fermentation of the lactose.
The litmus milk loses its color and becomes white.
Usually when a bacteria is capable of fermenting the lactose the medium will become pink because of acid production, however after enough time it will lose its color and become white.
Casein coagulation:
In certain cases the casein in the medium may form a curd.
If too much lactic acid is produced then the casein in the milk will become an acid curd. (curd is associated with acid production because of fermentation)
A casein curd may also form as a result of an alkaline reaction (rennet curd)
Casein Hydrolysis:
Casein hydrolysis usually occurs when the bacteria degrades casein instead.
The bacteria may fully hydrolyze the casein (proteolysis)
It may also partially hydrolyze it (purple color)
Litmus milk is an undefined/ differential medium.
Litmus milk being undefined means it is composed of complex ingredients rather than simple material.
A complex ingredient is made out of more than one thing in an unmeasured quantity, making it undefined.
If it were defined, we would know the exact amount of each ingredient.
Litmus milk is a differential medium.
A differential medium can distinguish between bacterial species.
In the case of the litmus milk medium, it can distinguish between:
lactose fermenters or nonfermenters.
Non-fermenters are most likely casein degraders.
Can degrade casein through hydrolysis.
Hydrolysis of casein is either complete or incomplete.
Partial casein hydrolysis results in a blue color because of ammonia production.
While complete casein hydrolysis will result in a translucent color.
The translucent color indicates the amino acids in the protein were completely broken down.
Oxidized litmus is purple
Chapter 16:
Sterilization refers to the complete elimination of vegetative bacterial cells.
Everything that is used in the laboratory must be sterilized.
Sterilization can be achieved through various methods:
Ionizing radiation
Gamma and x-rays
Ultraviolet radiation
Heat
Dry
Moist
Sterile filtration
Chemical
Ionizing radiation:
Ionizing radiation through the use of gamma and x-rays results in the formation of unstable ions which damage the cell.
This is done by forcing electrons out of their shell.
Ionzing radiation is used to clean disposable medical tools and equipment.
Benefits of ionizing radiation are that the item will not change temperatures during the process and that the item remains completely sealed.
Steam sterilization:
Moist heat sterilizartion. Usually done through the use of an autoclave.
The autoclave process occurs at 121 degrees celsius and a psi of 15 for 15 minutes.
Remember the ideal gas law: Pv=nRT which states that with the increase in pressure there will also be an increase in temperature.
It is also important to remember that with the increase in the size of the medium the amount of time should also increase.
Dry heat:
Dry heat is accomplished using a sterilization oven which requires high temperature for a long period of time.
Usually 180 degrees celsius for around 3 hours.
Sterile filtration:
Sometimes ingredients on the medium are heat sensitive so the use filtration method allows those ingredients to be individually sterilized.
Chemical filtration:
Formaldehyde can be used to clean glass ware. However, residue can affect results.
Chapter 17:
There are two forms of electromagnetic radiation used for the sterilization.
Ionizing
Ultra violet
UV-A: emmitted at wavelengths between 420-390 nm
UV causes indirect DNA damage by being absorbed by photosensitizers which then cause ROS damage
Photosensitizer proteins are activated because UVA is poorly absorbed, so they do not cause DNA photoaducts (damage to dna because of light)
UV-b 320-290
Absorbed by DNA and causes the formation of dimers.
Formation of dimers distorts dna polymerase creating single stranded sections on the dna.
UVC 280-200 nm
Absorbed by all cellular constituents. Howeverr most uvc is absorbed the atmospheric ozone and oxygen
DNA REPAIR:
Light repair
Dark repair
Light repair use photolyases enzyme to cut dimer covalent bond
Dark repair uses endonuclease enzyme to cut out damaged section and then fills it with dna polymerase and then combines it with DNA ligase.
Dark repair can occur in the dark and light.
Chapter 18:
Disinfectants: Chemicals used on non living surfaces
Antiseptics: chemicals applied onto living tissue.
Bactericidal agents/bacteriostatic:
Dependent on the amount used and time given.
Also not universal to all bacteria
Bactericidal agents: Kill bacteria by breaking down the peptidoglycan layer of the cell wall and cell membrane/
Bacteriostatic: Pause bacterial growth by disrupting protein synthesis and dna replication.
Decimal reduction time: amount of time needed to decrease the amount of bacteria by 90% on a surface
Chemothereaupetic agents: Antimicrobial chemicals that control bacterial population or growth within living tissue. Most are obtained from mold/ fungi
Can be natural or synthetic.
Streptomycin and penicillin – natural
Ampicillin and amoxicillin- synthetic
Kirby Bauer test: Utilizes mha plates, mueller hinton agar plates. Organism is swabbed over the entire surface to create a bacterial lawn then a fibrous disc impreghnated with specifgic amounts of antibiotics or anitmorbial agens are placed on surgace of agar and incubated.
MHA plate: mueller hinton agar plate contains acid hydrosylate, starrch and agar. The star allows the antibiotic/ antimicrobial to diffuse readily. Also protects from toxic components produced by bacteria
Zone Of inhibition:: Circular areas areoudn the disk where there is no growth or very little
Mimum inhibitory concentration: antimicrobial concentration near the outside region of the zoi
Antibiotics are chemotherapeutic agents not disinfectant/antiseptics
Chapter 19:
Naturally competent bacteria:
Naturally competent bacteria are able to take in genetic material from their surroundings without aid.
E.G: Streptococcus pneumonia and Subtilus bacillus
Although some bacteria are natrually competent the procedure occurs minimally therefore only some bacteria will grow on a minimal medium
Experiment:
A prototroph will grow on a rich and minimal medium
Auxotroph only on rich
Positive control (phe-) auxotrophs on LB
Negative control
Chapter 20:
Competency:
Competency is a term used to describe a bacterium’s ability to undergo transformation.
Transformation
The intake of genetic material from the environment.Naturally competent bacteria can undergo transformation naturally.
Incompetent bacteria are not able to undergo transformation naturally.
B. Subtilis is a species of bacteria that can absorb genetic material naturally. E. Coli is naturally incompetent and, therefore, is unable to.
Artificial Transformation:
Not all bacteria are physically capable of taking genetic material from the environment and implementing it into its own genetic material.
Why?
The primary inhibitor of transformation in incompetent bacteria is their cellular membrane. Remember, cellular membranes are selectively permeable; not everything will pass through them effectively. Incompetent bacteria have cellular membranes that do not allow genetic material to enter.
Therefore, if we want to use an incompetent bacteria for transformation, it must undergo artificial transformation.
Artificial transformation: When an incompetent bacteria absorbs genetic material from its environment after we disrupt its cellular membrane.
How?
SHOCK SHOCK SHOCK!!
Once again, remember how the primary inhibitor of transformation in incompetent bacteria is their cellular membrane? An effective cellular membrane can effectively select certain materials, therefore, if we want something that generally would not pass through the cellular membrane to enter it, we must disrupt the cellular membrane.
Methods of disruption (How to make an incompetent bacteria competent):
Ice cold CaCl2 bath.
CaCl2 at a concentration of 100mM makes the cell membrane more permeable.
Electroporation: Exposes the cell to high voltages and increases cellular membrane permeability.
DNA Insertion:
Transformation allows a bacterial cell to intake genetic material from the environment. Therefore, if we add genetic material to the medium a competent bacteria will be able to absorb the DNA (An incompetent bacteria which has undergone artificial transformation will also be able to).
What Genetic Material (type of DNA) was used?
Within the lab, plasmids were used.
Plasmids: dsDNA found within the cytoplasm of a bacterial cell. This DNA is separate from the circular chromosomal DNA and is able to replicate independently.
However, in most transformation experiments, raw DNA is not utilized. Vectors are.
Plasmid Cloning Vectors: Genetically engineered plasmids will most likely have some antibiotic resistance genes and exogenous DNA fragments.
Plasmid Cloning Vector= Antibiotic resistance gene + DNA you want to add.
pGLO plasmids:
beta-lactamase + (Several reporter genes including GFP)
Reporter genes:
Ori:
Origin of replication. Start of transcription.
Bla
Codes for beta-lactamase, antibiotic resistance.
GFP
Green fluorescent protein,
araC
Allows transcription of the set of genes.
***if not known***
A set of genes controlled by a singular transcription factor is known as an operon.
Remember how gene transcription is basically the coding for a specific gene that codes for a specific function/ characteristic.
When you do transcription normally you transcribe only a gene, but with operons your transcribing more than 1 gene, closely related genes.
So in this experiment you had plates with the
pGLO
The pGLO operon codes for the GFP protein
Amp
Antibiotic (ampicillin)
Ara
Arabinose *activates GFP*
Chapter 21:
Viruses are obligate intracellular parasites
They must be within another organism for them to replicate.
Viruses are simple organisms that consist of a protein coat with nucleic acid within it.
Viruses may have dsDNA, ssDNA, ssRNA, and dsRNA.
Viruses:
Lysogenic (temperate virus): The virus inserts its DNA into the host.
Lytic (virulent virus): The virus lyses the cell to escape.
How does a virus enter the cell:
Attachment
Penetration
Protein synthesis
Assembly
Release
Restriction length polymorphism:
RFLP
Fragments of DNA that are made by restriction enzymes are dependent on the recognition sites.
If you add a recognition site, you will reduce the fragment into 2 smaller fragments.
If you remove a fragment you will be combining two smaller fragments
Covid Virus (Lytic virus)
Herpesvirus (lysogenic and lytic)
Bacteriophage structure:
Capsid
Tail fibers
Tail sheath
Chapter 22 Fingerprinting:
DNA among everybody is 99% similar even when our DNA is compared to other species, genetic variation are polymorphism.
Most of human dna is exactly the same between individuals however regions of differences are known as polymorphism.
Restriction enzymes/endonucleases: Found in prokaryotic organisms, humans do not naturally have them. Cut dna at recognition sites.
Agarose gel is composed of three things: TAE buffer, agarose, ethidium bromide,
The DNA loading dye is added before the gel solidifies. This dye binds to the dna is and is visible under UV light. The DNA dye increases the density of the DNA sample so it weighs down in the well.
23 and 24:
Mannitol salt agar: contains NaCl peptones, beef extract, and mannitol
Chapter 23 & 24 PCR of Bacterial DNA
The Polymerase Chain Reaction (PCR) is used to exponentially amplify a target DNA sequence. Bacteria was grown on Mannitol Salt Agar: a differential and selective medium.
Mannitol Salt Agar: contains NaCl, peptones, beef extract, and mannitol. Differential because: Mannitol is the carbohydrate source, which may be fermented by S. Aureus and produce an acidic product. This acidic product would lower the pH of the medium and cause the phenol red pH indicator to turn yellow (below 6.8). If the bacteria cannot ferment mannitol, it will have to break down peptones or beef extract. This produces basic products like ammonia, which increases pH and turns the medium pink due to the phenol red pH indicator (above 7.4). If no reaction occurs, the pH stays neutral and is a red color. Selective because: it has a high salt concentration
PCR Steps:
⦁ Denaturation at 94℃ in which dsDNA was split into ssDNA
⦁ Annealing occurs between 50-65℃ in which a specific pair of oligonucleotide primers, usually 15-30 nucleotides in length, to bind to complementary sequences on the singlestranded DNA template.
⦁ Elongation occurs between 68-72℃ in which a thermostable DNA polymerase synthesizes the complementary strand to each ssDNA. (TAQ polymerase)
Master mix: dNTPs, loading dye (does not help PCR), primers, reaction buffer, magnesium
If you only see the DNA ladder, you might have forgotten to add your DNA template to the PCR reaction or the temperature could have been off in the thermal cycler.
Chapter 25 ELISA
ELISA: Enzyme-Linked Immunosorbent Assay used to detect antigen-antibody interactions (based on human body’s immune system)
Can be used to detect opiates and HIV in blood samples.
⦁ Direct ELISA: “SANDWICH ELISA” detects for a specific antigen to one antibody Wells of plate are coated with a specific antibody and a test antigen is added to each well. If the test antigen is recognized by the primary antibody, the antigen will be stuck in the well and the secondary enzyme, which is attached to an enzyme known as horse radish peroxidase is then attached to the primary antibody+antigen complex. Substrate is added that produces color when it reacts with the horse radish peroxidase.
⦁ YELLOW RESULT=CORRECT ANTIGEN
⦁ Indirect ELISA: detects for specific antibodies to one antigen Wells of the plate are coated with a specific antigen and then incubated with test antibody, secondary enzyme linked antibody is added which binds to test antibody that is bound to antigen, substrate is added that turns pink (positive)
⦁ PINK RESULT=CORRECT ANTIBODY
HORSE RADISH PEROXIDASE CATALYZES THE OXIDATION OF O-PHENYLENEDIAMINE BY USING HYDROGEN PEROXIDE AS THE OXIDIZING AGENT SO THAT IT GETS REDUCED. REDUCTION=COLOR CHANGE=POSITIVE RESULT!
B. Subtillis
M. Luteus
E.Coli