Lecture 1: What is Microbiology Flashcards
All life is either…
Prokaryotes or Eukaryotes
What do prokaryotes branch into?
Bacteria- unicellular
Archaea- unicellular
What do eukaryotes branch into?
Protists
- uni and multicellular(bc of evolution)
- genetically not cohesive group(don’t make sense as one group) and have been recategorized into different groups based on genetic similarities and differences
Fungi
-uni and multi cellular
plants and animals
- ALL multicellular
What are the benefits/ consequences of being multi vs unicellular
prokaryotes (bacteria and archaea) are unicellular (one cell constitutes whole organism) meaning if anything happens to the one cell its game over
plants and animals are multicellular so if you lost some cells(scraped off skin) it wouldn’t even matter because your made of so many cells
What is Microbiology?
The study of organisms(living) too small to be seen with the naked eye
• Bacteria, viruses, single celled eukaryotes
What microorganisms are visible to the naked eye?
Fungi and algae
microorganism
small and living
microbes
small
What microbes are multicellular?
Myxobacteria, slime molds
What is unique about viruses?
- They are non-living (but so small so they still need to be considered under the definition of mbio)
- don’t have the capacity to replicate unless host cell is there to provide important material
- not sustainable without a living cell
- can be referred to as microbes
What don’t microbes such as slime molds have?
- DONT have tissue diversification
- meaning none are differentiated such that their would be cells for detox and reproduction
- just made up of cells that are identical in function
- power in numbers
What is selective toxicity?
targeting foreign cells specifically while leaving host cells (your cells)
What are the challenges associated with infections caused by protists for example malaria vs infection caused by bacteria?
CAN’T USE an antibiotic that targets protist infections for bacterial infections
Issues with anti malaria drugs is you destroy your own cells to because eukaryotic cells are what your made of
Why can eukaryotic infections be challenging?
Because the type of cell infecting you is the same cell your made of called
- selective toxicity
- terrible selective toxicity
What techniques is microbio defined by?
• Culture media for isolation and growth of organisms in pure culture
• Biochemical to study cell components
- ex; poke a hole in membrane all intracellular components leakout and the you can use different techniques to sort and identify proteins
• Molecular and genetic techniques
- ex- these techniques were used to figure out protists aren’t a group of their own
culture
actively growing bacterial(or other living cells) sample
What is a mixed culture?
not pure
- many organisms
- also called polymicrobial
What is media/medium?
surrounding bacterial or microbial cells
- where they get nutrients from
- pulling nutrients from surrounding nutrient broth which is used to sustain growth and put waste in their
Can be liquid or solid medium
- ex; air around you ( take in oxygen contribute c02)
What is a pure culture?
- single species; just one type is there
If you swabbed your mouth and put it into liquid culture would that be a pure or mixed culture?
Mixed culture
-lots of different bacteria and organisms within; unsterile
Why Is Microbiology Important?
• Microbes are the oldest form of life
-> conditions of early earth were very hot no o2 just hydrogen was present archea) - allowed to self-replicate & form organic molecules in early earth (where it was forced to live without O2, & tolerate HIGH temps in order to evolve)
• Largest mass of living material on Earth
-> Organisms that live on you outnumber the eukaryotic cells that your made out of
• Carry out major processes for biogeochemical cycles
-> organisms are naturally forming a component where they fit)
• Can live in places unsuitable for other organisms
(@ -5 degrees will be very slow metabolic rate BUT still active, as well as -102 degrees to withstand folding, melting & DNA separation)
• Other life forms require microbes to survive.
- (cows and us cannot digest plant matter but cows can pack bacteria within their colon to digest cellulose via the bacterium - symbiotic relationship)
What happens when you suspect to have an infection?
You go to the dr and get antibiotics the antibiotics don’t know to keep good bacteria in the body and kill the bad bacteria they are just drugs so they clear out good bacteria that naturally exists (changes equ. biogeochemical cycle)
ex: vagina has lots of yeast & lactobacillus (prok. cell)
- b/c antibiotic target prok., you are taking for infection, you wipe out lots of bacteria in vagina - yeast (euk) that were not targeted, notice a lot of space now & extra nutrients, so the biogeochemical cycle in vagina is changed & the yeast start to overgrow, & now that needs to be treated
Every LIVING organism needs __
N2 is needed as nitrogenous bases and amino groups for amino acids
-> if you don’t have an adequate supply it can limit growth
What is the problem with N2 gas, even though N2 gas makes up ~78% of atmospheric gas?
TRIPLE COVALENT BOND is very stable
- & to disrupt this, we need LOTS of PRESSURE & TEMP that is impossible for a living cell to tolerate
- BUT they can use enzymes which require a lot of ATP- to perform nitrogen fixation
What is an example of a root nodule & what does it have?
Rhizobium spp.
- have enzymes to perform NITROGEN FIXATION - can disrupt cov. bonds 1 bond @ a time
What would happen then if Rhizobium spp was removed?
plant = nitrogen deficit
- & therefore, leaves colour & texture would be diff.
Which microorganisms are the oldest?
Earliest life form is archaea
from which unicellular then multicellular eukaryotes evolved
In a bacterium where are ribosomes located vs in a plant cell?
Bacterium will have 70s ribosomes in cytoplasm
plant cell will have 80s in cytoplasm AND 70s in mitochondria and chloroplast (bc of endosymbiotic theory)
What do all cells have in common?
- Cytoplasmic membrane
- Cytoplasm
- Ribosomes
- Genetic material
- Genome
- Chromosome
- Plasmid
Cytoplasmic membrane
Barrier that separates the inside of the cell from the outside environment
Cytoplasm
- Aqueous mixture of macromolecules(RNA DNA), ions (Na+ Cl- K+), and proteins
- consists of fluid and all components within
- very thick viscous like honey
Ribosomes
Site of protein synthesis
- key bc proteins are essential to function
made from protein
living cells need to have capacity
to make proteins
Genetic material
- All cells store their genetic information as DNA
* The information is divided into functional units called genes
Would viruses have all the features that are common among all cells?
NO
bc they are non living and acellular
DO NOT store genetic info as DNA BUT they could be DNA or RNA virus
Why don’t viruses fit in the following statement: “All cells store their genetic information as DNA”?
b/c viruses are ACELLULAR/non-living
& b/c viruses are diff. than living cells as some viruses are RNA viruses (flu, HIV, covid)
- NO pathway inside living cell that takes RNA & makes more of it
- therefore, RNA virus’s bring their OWN enzymes to let them make MORE RNA
- but those ENZYMES are ERROR-PRONE –> mutations/variations are a result (omricon, delta, HIV, flu etc.)
- therefore, NO vaccine for it as it is constantly CHANGING
Genome
A Cell’s Full Complement Of Genes (like a recipe)
- 46 chromosomes and mitochondrial DNA
like a collection of different recipe books
Chromosome
A genetic element carrying genes essential(for survival & cannot live without) to cellular function
- like a recipe book genes are the recipes the genome is a library of recipe books
- humans have 46 and bacteria only have 1
What happens if a organisms genes/ chromosomes were removed?
Day to day activity couldn’t happen bc there essential to function
Plasmid
A piece of DNA that carries non-essential genes(ex.Genes For Antibiotic resistance)
- small circular
Ex: penicillin resistance
- healthy person, doesn’t need to have a gene for penicillin resistance –> b/c no penicillin in them
- if they start taking penicillin –> they will become resistant to it
What are the 4 key points about a plasmid?
- May or may not be present
- If there, it provides some advantage
- Can replicate autonomously (even if rest of cell isn’t replicating, it can photocopy itself)
- Can pass their non-essential genes to other organisms to help them
If the plasmid is there, is it part of the genome?
If you had a bacteria, a culture media (bugs actively growing in this medium), & the medium doesn’t have any penicillin added, would this bacterium require a plasmid containing a gene for penicillin resistance?
NO because theres no penicillin there
- BUT if you add pen. to the growth medium (in ECF), the organism would benefit from having that recipe for penicillin resistance .
Describe the structure of eukaryotic cells
- eu= true karyon= nucleus
• Membrane bound nucleus- porous double membrane
• Membrane bound organelles- walls around everything that allows for compartmentalization
• Complex internal organization- (mitochondria has a mitochondrial matrix which is full of catabolic enzymes for breakdown it also has the inner mitochondrial membrane which has the ETC required for ATP production)
• Division by mitosis and meiosis.
What can you think of Eukaryotes as?
3-bedroom apartment (kitchen, living room etc. has DEFINED walls)
- everything is compartmentalized (longer to build)
- rent cost is higher
In eukaryotes is the highly organized interior entropically favourable or unfavourable? explain
a highly organized interior is entropically unfavorable because order costs energy but is necessary
What is the difference between mitosis and meiosis?
Mitosis; growth and repair; asexual; 2 identical daughter cells
meiosis; sexual reproduction; produces gametes
What are the Major groups of eukaryotic microbes?
Protists
- Protozoa
- Algae
- Slime molds and water molds
Fungi
Protists
unicellular or multi-cellular
no longer a cohesive group bc of new genetic info
without differentiation into tissues
- never have muscle, liver, epithelium tissue just have a number of cells that are similar so its just power in numebrs
Protozoa
animal-like microorganisms
Algae
photosynthetic plant-like microorganisms
- oxygenic photosynthesis
- precursor to plants
Slime molds and water molds
filamentous (important for absorption and extracellular digestion)
Fungi
Unicellular (yeasts), filamentous (molds), or multi-cellular (mushrooms).
What can you assume if you have a prokaryotic unicellular microbe and a unicellular eukaryotic microbe?
Eukaryotic will be BIGGER (need space to store everything)
Why do filamentous fungi (molds) get rotting?
b/c filamentous structure allows extracellular digestion & absorption of those nutrients that will facilitate fungal growth & you get a BIG SA to max absorption & benefit of the fungus
Describe the structure of Prokaryotes
pro= before karyon= nucleus
* 1st cells to come about in early aerobic earth
No membrane bound nucleus or organelles- single circular chromosome
Generally smaller (approx 1 μm diameter)
Simple internal structure
Divide by binary fission- asexual reproduction
Most are unicellular
What can you think of Prokaryotes as?
bachelor suite - STILL cook, go to bathroom, sleep, watch TV –> no functions lost
- simple, less organized
- cheaper to build
How do prokaryotes perform metabolism?
prokaryotes don’t have a mitochondria(no membrane bound organelles) but that doesn’t mean they can’t do metabolism they just do it all in cytoplasm, which is the site of all catabolic and anabolic reactions
Is the simple internal structure of prokaryotes entropically favourable or unfavourable?
More entropically favourable
- instead of producing membrane bound structures that have a higher level of organization b/c most is happening cytoplasmically
If a prokaryotic cell was given optimal conditions what would happen?
the cell could replicate in 10 mins (inexpensive)
- this is why food spoils
- for eukaryotes to do this it would take 10 hrs
What are the major groups of Prokaryotic microbes?
- Bacteria
- Archaea
- Viruses
Bacteria (eubacteria)
- Genetically diverse (different genes, different proteins, resulting in different metabolism)
- Extremely diverse metabolic styles (depending on where they live there will be different nutrients available so they need to be genetically diverse
- Includes both pathogens and non-pathogens (could become pathogenic if they pick up a different gene, ex; covid variant)
Archaea (archaebacteria)
- Genetically and biochemically distinct from bacteria
- Also have diverse metabolism (produce different waste)
- Never pathogenic (doesn’t mean they cant become pathogenic tmr,ex of genetic plasticity)
- Most famous for living in extreme environments (we have them in our gut)
Pathogens
cause disease
What does it mean to be genetically plastic?
lots of change to genetics that can occur
* we are to complex to do this
Why is it beneficial for archaea to live in extreme environments?
less competition for resources
dominante environment
What makes archaea able to live in extreme environments?
- have MODIFICATIONS (“extra clothes” that make them more able to handle/resistant)
- proteins that can stay folded in extreme temps
- more C-G bonds
Viruses
- CAN BE CALLED MICROBES BUT NOT MICROORGANISMS
Acellular infectious particles (NO plasma membrane, protein spikes to permit interaction with host cells, protein compartment to store genetic info)
Extremely small (small genome, efficient)
Obligate(obligation) intracellular (be inside of a cell that provides it things it needs but it doesn’t have) parasites (+/- relationship)
Lack independent metabolism
• No ribosomes
• No ribosomal RNA
• Cannot be classified with other microbes.
*VERY EFFICIENT –> ONLY BRING WHAT HOST CELL DOESN’T HAVE
Which one is the plus and the minus in a paarsitic relationship?
virus= +
host cell= -
Viruses have no rRNA so what does this mean in terms of how we can classify them?
we use sequences of genes that encode rRNA to classify living organisms to particular genus and species=> viruses don’t have this so we can’t classify them like this
Give an analogy explaining how a virus is an intracellular parasite
Intracellular- If you go to someone’s house and use their stove, shower this means that, that persons bill goes up and they cant cook on their stove because you are using it
= parasitising and draining resources -> making a person tired
* viruses are selfish
Describe Evolution and Diversity of Microbial Cells
• First anaerobic life appeared between 3.8 and 3.9 billion years ago
* conditions of early earth would’ve allowed for production of organic molecules eventually come up with cellular structure that started replication
• Photosynthetic bacteria (cyanobacteria) oxygenated the Earth about 2 billion years ago
- Allowed the evolution of modern eukaryotic microorganisms (aerobic, energy yield is increased)
• First plants (like cyanobacteria but larger and more complex) and animals appeared about 0.5 billion years ago
What’s the diff generally in terms of energy yield from an organism that’s doing anaerobic metabolism (ferm. or respir.) & an organism that’s doing aerobic metabolism?
A lot less energy in anaerobic metabolism
Luca
lowest universal common ancestor
Describe the phylogenetic tree with Luca
In the phylogenetic tree Archaea look closely related to Eukarya
- > shows that genetically eukarya and archaea are closely related
- > simple prokaryotic structure doesn’t say anything about genetic relationship
How can organisms be classified?
By comparing small subunit (SSU) rRNA genes
Describe ribosomes in prokaryotes
70S ribosomes
- large subunit= 50 s
- small subunit= 80 s
• 16S SSU rRNA
What are ribosomes made out of?
Protein and rRNA
Describe ribosomes in eukaryotes
80S ribosomes
- large subunit= 60s
-small subunit= 40s
• 18S SSU rRNA
16S SSU rRNA
piece of rRNA made by transcription (IN PROKARYOTES)
16s= size
- this sequence is what we look at to sequence the gene to look for relationships between organisms and put them into species
(what gets sequenced to establish relationships)
What is rRNA?
structural detail to make ribosomes made by transcription
18S SSU rRNA
- IN EUKARYOTES
- gene sequence to establish relatedness of eukaryotes
What are the differences between eukaryotic and prokaryotic ribosomes?
both have the same function
and are not additive
*only difference between the 2 is size, eukaryotic ribosome is bigger than prokaryotic ribosome
What is a difference between the 18S SSU rRNA and 16S SSU rRNA?
The 18S SSU rRNA is longer than prokaryotic rRNA (made up of more nucleotides)
18 pieces of rRNA that make up the ssu vs 16 pieces that make up in prokaryotes
What is S?
Svedburg unit
- measurement of sedimentation
Why do we call the ribosomes 80s vs 70s?
If you were to put the ribosomes (70s, 80) and sub units (50s, 30s, 60s, 40s) and apply centrifugal forces the heavier stuff goes to the bottom
-> separate according to density thats why you assign the numbers bc 50s is heavier vs 40s
Why do we look at the SSu rRNA genes?
rRNA genes change slowly over time which is useful to evaluate long lasting relationships
*if we look at it today its helpful for us to see relatedness that might be very long lasting
Examines genetic differences rather than morphological differences
Summary of rRNA sequencing
1) isolate DNA from each organism
2) Make copies of rRNA gene by PCR
3) Sequence DNA
4) Analyze sequence
- align the rRNA sequences and see which nucleotides are different the sequence with the most nucleotides similar are closely related
5) generate phylogenetic tree
What are the basic steps involved in sequencing rRNA genes
Step 1: DNA is collected from a pure culture(one species one strain)
Step 2: The SSU rRNA gene (DNA) is amplified using the polymerase chain
reaction (PCR)
- amplify so you have many pieces
Step 3: The gene is sequenced
Step 4: Sequence is aligned with sequences from other organisms
• Number of differences is used to calculate evolutionary distance
-> % sequence homology (what % of the sequences are the same)
What is PCR?
a technique used to synthesize many identical copies of a short sequence
of DNA
Phylogenetic tree
A graphic representation of the evolutionary distance between organisms.
What are Phylogenetic trees based on?
Phylogenetic tree based on 16S or 18S ribosomal DNA sequences
All organisms can be grouped into 3 distinct domains of life: Bacteria,Archaea and Eukarya
Microorganisms are_________than ___and___
Why?
Microorganisms are far more genetically diverse than plants and animals
- microorganisms are very small so they are able to make a lot of change to their genetic material
- > referred to as genetically plastic because constantly mixing and matching genetics coming up with new genotypes which allows them to acquire antibiotic resistance
- PLANTS AND ANIMALS ARE TO COMPLEX TO BE GENETICALLY PLASTIC
Bacteria & archea are both proks but…
Genetically Archaea and Bacteria form different branches
-> have enough genetic diversity despite cell structure homology
species
is the fundamental unit of biological diversity
Biological species concept
sexually reproducing organisms
sperm and egg come together, produce genetic variation and through mitosis become new organisms
what is a species?
Phylogenetic species concept:
• “A group of strains that share certain (NOT ALL) diagnostic traits, are genetically cohesive and have a unique recent common ancestor”(We look at genes that change slowly so we anticipate they have evolved together from common ancestor)
Biological species concept DOESN’T work for what?
For bacteria the biological concept of species doesn’t work because they reproduce asexually (duplicate contents) but nothing new is created
In practice, species of Bacteria and Archaea should have:
• Most (but not all) characteristics in common
=> very genetically similar but NOT identical
• Greater than 97% sequence similarity in the 16S rRNA gene
• High degree of genome similarity
- DNA-DNA hybridization
=> the 2 DNA strands should be hydrogen bonding bc they should be complementary
• In the very near future:whole genome sequences?
=> we look at one rn bc its cheap but if we can look at whole genome it will be better
If 2 bacteria have 96% sequence similarity in their 16S rRNA gene will they be same or diff. species?
Different species bc <97%
Describe the process of DNA-DNA hybridization
take DNA from one organism your comparing
heat it up to break the hydrogen bonding to get a single strand and do the same to the other genome
so you get 2 single stranded DNA strands from 2 diff cells
take the complementary strands and if they are similar the DNA should hybridize (the 2 hybridized strands will be mostly complementary but not all of it will be complementary)
How do Bacteria and Archaea reproduce?
reproduce asexually
- no change no genetic variation
If they have >97% sequence similarity in the 16S rRNA gene, is that 100%/are they identical?
No
How do microbiologists name organisms?
Microbiologists use Hierarchical classification (big umbrella small subgroups)
• Groups of organisms are placed in successively larger groups
• In practice: Species, genus and phylum are commonly used
What is the Taxonomic hierarchy?
Domain Phylum Class Order Family Genus Species
Greater than 97% similarity= members of 1 species
then we divide the 1 species into different strains according to the differences that make them not identical
Opportunistic pathogen
waits for opportunity
pathogen= can cause disease under right circumstances
What is interesting of warm bloodied animals like cows & us in terms of e.coli?
E.coli is beneficial in large intestine in humans but harmful in other places
cows also have e.coli but it is not the same strain
-> SAME SPECIES BUT NOT THE SAME STRAIN(>97% SO CALLED SAME SPECIES BUT NOT 100% IDENTICAL SO DIFF STRAIN)
Describe the binomial species names
Escherichia (genus name) coli (species name)
Genus (capitalized, like last name; smith lots of ppl have that last name)
Specific epithet (not capitalized, like 1st name, makes individual unique)
Strains can be identified by symbols after the species name ex. E. coli K12
What are the rules for classification & nomenclature?
- Names are latinized
- Italicized Or Underlined
- Genus Capitalized,epithet is not
- Genus Name Maybe Abbreviated The Second Time It’s Used:E.coli
- Trivial Names (nick names) can be used,but not follow the rules
One genus_____
can have many or just 1 species
What should the strain look like in terms of classification & nomenclature?
written like normal no underlining or italicizing
Who was Robert Hooke?
The first to describe microbes
• Used a compound light microscope – uses 2 lenses to magnify the image (light bulb is used to magnify the image)
Allowed magnification up to 30x
• Used it to observe:
• Cells in cork (cork= from a tree, so a plant cell which is eukaryotic and large cell so it isnt hard to see prokaryotic cell would’ve been hard to see)
• Bread mold filaments – 1st microbe
• Beginning Cell Theory–all living things are composed of cells
How did viruses 1st get discovered?
1935; looked at tobacco plants there were diseased
- looked under microscope but nothing there
1935: 1st electron microscope looked at plant and saw 1st virus. tobacco mosaic virus (TMS)
- this was to small to be seen under light microscope
Who was Antoni van Leeuwenhoek?
Built microscopes that magnified specimen by 50-300x (allowed him to see prokaryotes)
Observed single celled microorganisms – called them “animalcules”
• First discovery of bacteria
What did Louis Pasteur discover?
Studied wine and beer production
• Yeasts convert sugar to alcohol in the absence of oxygen (anaerobic)
- Fermentation – “La vie sans air”
• Bacteria can sour wine by converting alcohols to acid (changes functional group to carboxylic acid which will protonate lower pH)
• Developed a method of gentle heating to kill unwanted bacteria – Pasteurization
Fermentation
“La vie sans air” (life without air)
- > only 2 ATP/ glc molecule which is an disadvantage
- If o2 is available they will use aerobic metabolism= 32ATP
Pasteurization
lowers the microbial count in order to protect against disease
- take milk and subject it to a mild heat treatment which will decrease microbial count
Describe how milk gets pasteurized
milk carton has many bacteria but bc its pasteurized there’s less bacteria and the disease causing bacteria have been killed and stored at room temp it slows the growth of organisms
What will happen if you brought pasteurized milk home & forgot to put it in the fridge for 5-6 hours & left it on the counter? What will happen the next day?
You would see curdling
-> clumps of bacteria bc heat will kill them and destroy their structure so they will aggregate early on all their properties will be ruined bc of microbial growth
What was Louis Pasteurs procedure for spontaneous generation?
Prepared meat infusions inside of long swan-necked flasks Boiled the infusion to sterilize it
As long as the flask remains upright, dust and microbes cannot enter, and the infusion remains sterile
What did Louis Pasteur’s spontaneous generation experiment lead to?
Led to the development methods for controlling the growth of microorganisms (aseptic technique)
What is aseptic technique?
minimize contamination
- minimize likelihood of microbial growth
spontaneous generation
things just come about
Describe in detail, Louis Pasteur’s spontaneous generation experiment
a) Non Sterile liquid is poured into a flask (broth is dirty, contaminants bacteria end up at the bottom of the flask)
(the nutrient broth that was poured in had carbon source, nitrogen source iron, all the things needed for most organisms to grow)
- neck of the flask is drawn on in flame (is drawn out so that things can’t drop into flask, anything that did drop in is settled, this allows for a controlled experiment because your controlling entry of new microorganisms that aren’t already there
- liquid is sterilized by extensive heating (killed all bacteria that were deposited early on, setting to point of zero)
steam forced out open end
b) liquid cooled slowly
- dust and microorganisms trapped in bend of flask
long time goes by
liquid remains sterile indefinitely (flask has open end)
(NOTHING IN THERE THAT PROVIDED REPRODUCTIVE CAPACITY FOR NEW CELL TO COME ABOUT)
c) flask is tipped such that microorganisms- laden dust contacts sterilized liquid
short time goes by
liquid putrefies
=> Introduced bugs that were settled in neck of the flask into broth (liquid became cloudy and smelly)
INDICATES NUTRIENT MEDIUM COULD SUPPORT GROWTH MEDIUM (initially there were no organisms in there but as soon as you put in new cells can come)
THIS DISAPPROVED SPONTANEOUS GENERATION
What did Louis Pasteur want to know?
Wanted to know if spontaneous generation is possible
-> If you left the liquid knowing there’s nothing living present in broth, will new microorganisms be able to spontaneously arise given they have this nutrient broth
THE ANSWER HE GOT WAS NO
sterile
clear no smell
What did Robert Koch study?
Studied anthrax – responsible for epidemics in livestock
- livestock get infected spread it to other organisms living close together
How did Robert Koch carry out his experiment?
He isolated bacteria from the carcass of a diseased animal – Bacillus anthracis
• Injected healthy animals with the bacterium
• Animals became ill with anthrax
-supported evidence of his hypothesis that bacillus anthracis were responsible for anthrax
• Re-isolated B. anthracis from the test subjects and showed that it was identical to the one from the cracus of the diseased animal
• Established a set of criteria for relating a specific microbe to a disease
Koch’s postulates
set of criteria for relating a specific microbe to a disease
What are the 4 Koch’s Postulates?
- The suspected pathogen must be present in all cases of the disease and absent from healthy animals (microscope staining)
- The suspected pathogen must be grown in pure culture (Lab cultures)
- Cells from a pure culture of the suspected pathogen must cause disease in a healthy animal (experimental animals)
- The suspected pathogen must be reisolated and shown to be the same as the original (lab resiolation and culture)
=> isolating for a 2nd time in pure culture allows us to make sure that the organism above didn’t die from other causes but from the organism that was put in him
What are the experimental aspects of Koch’s Postulates?
Diseased animal
- observe blood/ tissue under the microscope (the dish in not a pure culture you have red blood cells and suspected pathogen)
- streak agar plate with sample from either a diseased or healthy animal (in diseased there will colonies of the suspected pathogen)
- inoculate healthy animals with cells of suspected pathogen
- remove blood or tissue sample and observe by microscopy
- pure culture must be same organism as before
healthy animal
- will have pure culture (just red blood cells)
- in the streak agar plate there will be no organisms present)
If you pretend that TB was new & people are showing signs/symptoms. then you isolate from many of them. microbacterium TB (bug that causes TB). But if you had 100 people that showed same signs/symptoms & you isolate microb. TB from 75 of those but 25 don’t have microb. TB in their lung at all. What is problem with your hypothesis that microb. TB is responsible for causing TB?
It could be something else bc what abt the other 25 ppl
What did Robert Koch realize?
Realized that solid media provided a simple way to obtain pure cultures
Describe the solid media
Broth medium (liquid medium) solidified with agar
What is agar?
- Polysaccharide derived from marine algae
• Melts at ~ 97°C and polymerizes (solidifies) at ~ 43°C
• Cannot be degraded by most microorganisms (similar to how we can’t degrade cellulose, so remains a solid support) - powder added to liquid medium then poured into empty petri dish once it cools it solidifies and provides a solid growth medium
Typical Petri plate =
nutrient broth medium + 1.5% agar
Solid growth medium vs. liquid growth medium
in a solid growth medium you can transfer bacteria to another plate to create a pure culture
in liquid growth medium you can’t pick one and create a pure culture because its a soup there everywhere
If you had a recipe with peptone, beef extract, NaCl, and agar would it be true or false to consider it a liquid growth medium?
False
because it contains agar which makes it a solid medium
If you had a recipe with peptone, beef extract, NaCl would it be true or false to consider it a solid growth medium?
False
because there’s no agar
Why is agar used?
If it wasn’t for agar it would be a liquid growth medium and you wouldn’t be able to get a pure culture
Describe the nutrient broth medium
Peptone- protein digest
Beef extract- emulsification of beef tissue which will have lipids nutrients to support microbial growth
NaCl- establish concentration gradient
Agar- solidifying agent
What is the streak plate technique?
• One edge of a plate is inoculated with a concentrated sample of bacteria
• Sample is diluted by streaking it across the surface of the plate
- To deposit individual cells on the plate (separate from other cells)
• Plate is incubated
• Individual cells grow to form colonies (that can be seen with the visible eye)
• Can be used to create a pure culture
What is a potential source of error? Is there a way to guarantee if just 1 cell fell into this spot to that colony?
you might have had 2 cells which then duplicated which is a source of error
Why is the loop flamed and what would happen if we don’t let it cool?
The loop is flamed to set microbial count to zero and if you don’t let loop cool you kill microbes so no viable growth
Colony
a mass of cells that (ideally) arose from one single cell
- cells come from a single cell that fell in one location on the plate
What is the optimal temp for growth?
37 degrees celcius
Explain the streak plate technique
What you smear on the plate is highly concentrated so you flame the loop to set microbial count to zero again wait for it to cool then drag 1 way and dilute then sterilize again
- by constantly dragging your diluting and counts become very low in comparison with what you started with and plate will look like there’s nothing on it
Spread Plate and Pour Plate Techniques
• Sample is diluted before plating
• Diluted sample can be spread over the
surface of the plate with a sterile spreader
• Separate cells grow into colonies on the surface of the plate (one cell= one colony)
• Or can be mixed with molten agar (~ 45°C, bc any higher the agar will harden)
- pour it so cool enough it won’t kill organisms
• Colonies form embedded inside the plate
If you put into incubator for 4 days at 37 what would happen?
There will be over growth
-> colonies run out of food and die bc of excessive binary fission
What would be the problem if you put in boiling agar?
You would kill the bacteria
Compare how the colonies grow in the spread plate vs pour plate techniques
in spread plate growth is only on top
on spread plate get multi dimensional separation (top, middle bottom)
What does a cooler temp mean?
slower growth
The Standard Plate Count
Spread and pour plates allow you to calculate the concentration of bacteria in a population (bacterial titre)
titre = # colonies/ (volume)(dilution)
• titre is expressed in cfu/ml
• cfu = colony forming unit
When can a standard plate count be done?
Done in hospitals
- > worried you have an infection , blood gets taken and they try and grow bacteria
- > nothing should grow
- > if there is something there’s been contamination or there’s an organism in your blood
serial dilution
important its done at the same time bc you want to count organisms and they will keep growing
-> control
Countable plates
We normally count plates with between 30 – 300 colonies < 30 – not statistically significant
> 300 – colonies grow into each other – inaccurate counts
When we have more than one countable plate…
• Calculate titre from each and take the average.
In theory how should the plates be?
Have the same but bc of error that doesnt happen
Does the standard plate count living or dead bacteria or both?
Living bc dead would not be able to grow
-. called a viable count
