Lecture #1 - What is Microbiology? Flashcards
Which organisms are too small to be seen with the naked eye?
Bacteria, viruses, single celled eukaryotes
What is different about viruses?
NON-LIVING
- not sustainable without a living cell, can’t replicate unless the cell is there to provide imp. material
- therefore, don’t fit under the umbrella of living organisms
ALL LIFE (if it’s living) is either…
Prokaryote cells or eukaryotic cells
What does prokaryote cells branch into?
BACTERIA - unicellular
ARCHEA
What does eukaryotic cells branch into?
- Protists - uni & multicellular (don’t make sense as 1 group genetically)
- Fungi - uni & multi
- Plants - all multi
- Animals - all multi
What is the consequence of being unicellular over multi?
just 1 cell that constitutes the ENTIRE living organism
- therefore, if it is destroyed/denatured which affects it’s DNA structure, then it’s done
WHEREAS, multi don’t notice if some of their cells are scraped off (no impact on their ability to survive, physio, etc.)
Which microorganisms are visible to the naked eye?
Fungi, algae
Which microbes are multicellular?
Myxobacteria, slime molds
What is a result of microbes that are multicellular, such as Myxobacteria, slime molds?
DO NOT have tissue diversification
- made up of 1 or more cells who’s function is identical
- therefore, has power in #’s only
What is 1 reason why Euk. & fungal infections can be challenging?
b/c the type of cell that is infecting you is the same type of cell that is making you up –> CAN’T use antibiotic (hard to distinguish what to kill & what not to)
–> terrible selective toxicity
WHEREAS, for bacterial infections, you CAN use an antibiotic & it targets a prokar. cell, you don’t have to worry about euk cells typically
Selective toxicity
targeting foreign cells specifically while leaving host cells (your cells) unharmed
What techniques is microbio defined by?
- Culture media (LIQ OR SOLID!) for isolation and growth (to INCREASE their #) of organisms in pure culture
- Biochemical methods to study cell components (to ISOLATE, IDENTIFY, & make more SENSE of it)
- Molecular and genetic techniques (ex: protists don’t make sense to all fit in a group due to GENETIC TECH, we can use this to see what is related to each other in order to organize)
What is culture?
culture = actively growing bacterial sample (or other living cells)
What is media/medium?
what is immediately surrounding the microbial or bacterial cells for ex, & is where the bacteria gets their nutrients from
- for us, ours can be air surrounding us
- pulling nutrients from surrounding nutrient broth
- then put their wastes into that medium
What is a pure culture?
specifically a single strain/single species
- just 1 type is there
What is an example of a mixed/polymycrobrial culture?
mouth swap when it is est. on a medium b/c we breath air & eat food (LOTS of diff bacteria/organisms within –> unsterile)
Why Is Microbiology Important?
- MICROBES are the OLDEST form of life (MICROBES (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 (out#’s US, b/c organisms in gut, skin, vagina etc. are higher in #, climate change adds selective pressure as to what will evolve)
- 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 cellulse VIA the bacterium - symbiotic relationship)
What is the conseq. of an antibiotic drug which kills according to its mode of action?
it can kill GOOD bacteria too
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
What happens after you kill off bacteria in the vagina?
chemical change will become MORE NEUTRAL (lost lactic acid source) & @ risk for sexually transmitted infection
Root nodules are full of…
BACTERIA
Every LIVING organism needs _____
N2 gas
- if they don’t have in adequate amount, it will 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
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. (perform nitrogen fixation) was REMOVED?
plant = nitrogen deficit
- & therefore, leaves colour & texture would be diff.
All cells have the following (7) in common:
- Cytoplasmic membrane
- Cytoplasm
- Ribosomes
- Genetic material
- Genome
- Chromosome
- Plasmid
Why do VIRUSES NOT have the same common structures as ALL cells?
viruses = NON-living/Acellular (WITHOUT CELLS)
Cytoplasmic membrane
Barrier that separates the inside of the cell from the outside environment
Cytoplasm
Aqueous mixture of macromolecules, ions, and proteins
- fluid & all components within = cytoplasm
- THICK viscosity
Ribosomes
Site of protein synthesis
- key to any living cell b/c proteins are essential for function
Genetic material
• ALL cells store their genetic information as DNA
- think: books in house written in Eng (DNA), but could be written in German (RNA)
• The information is divided into functional units called genes (contain recipe/info to make a protein)
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
46 genomes (recipe books) collectively
Chromosome
A genetic element carrying genes ESSENTIAL (for survival & cannot live without) to cellular function
Bacteria has only 1 chromosome, HUMANS have 46
Plasmid
A piece of DNA that carries NON-essential genes (ex. Genes for antibiotic resistance)
- can help but may not be needed
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 of 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?
YES - b/c it’s a gene made out of DNA, located in the inside of the cell
- but don’t need to be there
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 pen. resis?
NO - b/c no pen. there
- BUT if you add pen. to the growth medium (in ECF), the organism would benefit from having that recipe for pen. resis.
What are the 2 structural categorization of microbes?
- Eukaryotes
2. Prokaryotes
Eukaryotes structural categorization
- Membrane bound nucleus
- Membrane bound organelles
- Complex internal organization
- Division by mitosis and meiosis.
What does membrane bound mean?
place walls around so you can compartmentalize
see in this Eukaryotes
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
Is organization fav. typically?
NO - costs a lot of energy to maintain - entropically unfav. but necessary to function
(we see this in Eukaryotes)
Difference b/t mitosis & meiosis
mitosis - growth/repair (asexual repro)
meiosis - sexual repro (produces gametes)
What is a general rule b/t a Euk. microbe & a prok. microbe, even if both are unicellular?
EUKARYOTE will be BIGGER!
- need space to store everything
What are the major groups of eukaryotic microbes?
- Protists
- Fungi
Protists
unicellular or multi-cellular WITHOUT differentiation into tissues (just power in #’s)
- no longer a cohesive group
What are the 3 types of protists?
- Protozoa
- Algae
- Slime molds and water molds
Protozoa
animal-like microorganisms
Algae
photosynthetic plant-like microorganisms
- capable of oxygenic photosynthesis –> making oxygen that we can use (we’re aerobic)
Slime molds and water molds
filamentous
- long & thin
- imp. for absorption
Fungi
Unicellular (yeasts), filamentous (molds), or multi-cellular (mushrooms).
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
Prokaryotes
- No membrane bound nucleus or organelles
- Generally smaller (approx 1 μm diameter)
- Simple internal structure
- Divide by binary fission (asexual)
- 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
- if you give it a set of optimal conditions that cell maybe replicates in like 10 mins only (whereas our cells take over 10 hours)
Since there is NO membrane bound nucleus or organelles in Prokaryotes, where does metabolism take place for ex?
does metabolism in the OPEN b/c no mitochondria
- CYTOPLASM will be cellular location for all the catabolic & anabolic rxns (does all same things)
Is the simple internal structure of prokaryotes entropically fav or unfav?
more entropically FAV - instead of producing membrane bound structures that have a higher level of organization b/c most is happening cytoplasmically
What are the major groups of Prokaryotic microbes?
- Bacteria
- Archaea
- Viruses
Bacteria (eubacteria)
• Genetically diverse
- translates into diff. genes/recipes, which translate into diff. recipes (meaning diff. metabolism)
- Ex: won’t get drunk from running
• Extremely diverse metabolic styles
- depending on where they’re living, diff nutrients may be avail or unavail
• Includes both pathogens and non-pathogens
Pathogens
causes disease
- has something to make you sick
Why does bacteria include both pathogens & non-pathogens?
b/c they are fairly giving & experimental with genetic material in their environ.
- means: non-pathogenic CAN change to a pathogen if they pick up a certain gene
- same with variants (some more/less transmissible)
Archaea (archaebacteria)
• Genetically and biochemically distinct from bacteria
- but also have prok. cell structure
• Also have diverse metabolism
- b/c their genetics are diff.
• Never pathogenic
- but CAN change tomorrow for ex b/c of genetic plasticity
• Most famous for living in extreme environments.
Genetically plastic
lots of CHANGE to genetics that can occur
What makes archaea able to live in extreme environments? What is the benefit of this?
- can also leave in our gut (not extreme)
- have MODIFICATIONS (“extra clothes” that make them more able to handle/resistant)
BENEFIT: dominant, much less competition for the resources
Viruses
- Acellular infectious particles (have protein compartment that stores genetic material, protein spikes on external survive to permit interaction with host cell)
- Extremely small –> efficient ONLY BRING WHAT THE HOST DOESN’T HAVE (selfish)
- Obligate intracellular parasites (obligation to be inside of a cell that provides things it doesn’t have on it’s own but needs, & parasite is a +/- relationship (virus +, host -)
- Lack independent metabolism
Viruses lack independent metabolism, which means…
- No ribosomes
- No ribosomal RNA (we use sequence of genes that encode rRNA to classify living organisms to a particular geneis & species)
- Cannot be classified with other microbes.
Describe Evolution and Diversity of Microbial Cells
• First anaerobic life appeared between 3.8 and 3.9 billion years ago (early earth had no O2 in atmosphere)
- Photosynthetic bacteria oxygenated the Earth about 2 billion years ago (as a result of CYANOBACTERIA)
- Allowed the evolution of modern eukaryotic microorganisms.
• First plants and animals appeared about 0.5 billion years ago
- multi, complex
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?
LOT LESS ENERGY when doing ANAEROBIC metabolism
Luca
LOWEST universal common ancestor
Describe the phylogenetic tree with Luca
Prokaryotes are separate from each other, showing:
- genetically, closer relationship of archea to eukarya, than archea to bacteria
- simple prok. structure clearly doesn’t mean much about genetic relationship Ex: being brunette doesn’t say anything about intellect etc.
Describe ribosomes in prokaryotes
– 70S ribosomes
• 16S SSU rRNA
LSU = 50S SSU = 30S
- protein synthesizing organelle
- made out of protein & rRNA
Describe ribosomes in eukaryotes
– 80S ribosomes
• 18S SSU rRNA
- LSU = 60S
- SSU = 40S
- protein synthesizing organelle
- made out of protein rRNA
Why is the ribosome subunits not additive?
b/c when they come together they form unique bonds that cause the structure to be slightly smaller when assembled together
- think: knuckles together
What are small subunit (SSU) rRNA genes?
piece of RNA made by transcription
- # infront of it is characteristic of the size
Which SSU is larger between prokaryotes & eukaryotes & why?
prokaryotes: 16 SSU rRNA is made up of FEWER nucleotides (shorter)
eukaryotes: 18S SSU rRNA is made up MORE nucleotides (larger)
What do we use the SSU rRNA gene for?
to sequence the gene or look for relationships b/t organisms
- to put organisms into diff. species
What does the 16S or 18S SSU rRNA do?
gene sequence to establish relatedness of euks or proks depending which one
What happens when we apply centrifugal force to the tube with pieces of prok. & euk ribo, completely assembled ribo’s & free SSU & LSU’s?
HEAVIER stuff go to bottom
- separate out according to their density!
S = Sredberg unit is…
measurement for the RATE of sedimentation
Comparing small subunit (SSU) rRNA genes
- rRNA genes change slowly over time
* Examines genetic differences rather than morphological differences
Multiple proteins & pieces of____ per structure
rRNA
Compare & contrast Euk & Prok ribosome:
- exactly same functions
- Euk is larger
- also not additive for same reason
- both have piece of rRNA (Euk is bigger - 18S)
What is the reason why we look @ 16S or 18S (depen. on which)?
b/c gene changes VERY SLOWLY
- therefore, same for a long time (helpful to see relationships that can be long lasting
- beneficial for us to use as they can give genetic diff’s which is better (than morphological - ex: shape; physical dets, doesn’t have anything to do with what true relationship might be)
What would be the ribosome in a bacterium (prok. cell)?
70S ribosome
What would be the ribosome in a Euk. cell (plant cell)?
80S ribosome
What is imp. in terms of ribosomes in Euk cells? What about in a plant cell specifically (euk cell)?
Euk’s have 70S ribosome inside the mitochondria (according to endosymbiotic theory)
if plant cell, it will also have a 70S ribosome in its chloroplast
What are we looking for when we are looking at an analyzed sequence?
look for homology & where they are diff
- will have a closer relationship if they have more nuc sequence homology
Basic steps involved in sequencing rRNA genes
Step 1: DNA is collected from a pure culture
Step 2: The SSU rRNA gene (MATERIAL IS DNA) is amplified using the polymerase chain reaction (PCR)
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
- looking for % sequence homology
Polymerase chain reaction (PCR)
a technique used to synthesize many identical copies of a short sequence of DNA
Why do we use PCR?
so we have multiple copies to sequence efficiently & accurately
What are we looking for when we sequence rRNA genes?
% sequence homology
Phylogenetic tree
graphic representation of the evolutionary distance between organisms. (where they are relative to Luca)
5 steps of sequencing rRNA genes SUMMED UP
- Isolate DNA from each organism
- Make copies of rRNA gene by PCR
- Sequence DNA
- Analyze sequence
- Generate phylogenetic tree
What is the Phylogenetic tree based on?
Phylogenetic tree based on 16S (proks) or 18S (euks) ribosomal DNA sequences
Tree of Life
All organisms can be grouped into 3 distinct domains of life: Bacteria, Archaea and Eukarya
as a result of the sequences
What are microorganisms compared to plants & animals?
Microorganisms are far more genetically diverse than plants and animals.
Bacteria & archea are both proks but…
on separate branches, despite homology & cell structure (obvi doesn’t mean much) as we can see from the tree of life
What is the reason for, “Microorganisms are far more genetically diverse than plants and animals?”
b/c they’re VERY SMALL & simple (get away with it better compared to humans that are complex for ex; would experience serious consequences from changes to genetic material)
- able to make a lot of changes to their genetic material
- call them PLASTIC as they are coming up with new PHENOTYPES all the time
- therefore a lot of genetic diversity from this change
- constantly adding more in, ex: acquiring antibiotic resis, new toxins, new phenotypes having capacity to cause a lot of change within them
The species is the fundamental unit of _________
biological diversity
Biological species concept
- for sexually repro. organisms
- says, members of the same species, must be able to breed with one another & then the offspring they produce must be fertile
Biological species concept DOESN’T work for what?
BACTERIA
- b/c they reproduce entirely ASEXUALLY (duplicate their content)
Phylogenetic species concept:
“A group of strains that share CERTAIN diagnostic traits, are genetically cohesive and have a unique recent common ancestor”
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 (looking at this gene)
- High degree of genome similarity (would expect this even though we are just looking at 1 gene)
- DNA-DNA hybridization
- In the very near future: whole genome sequences?
If it was 96% sequence similarity in their 16S rRNA gene? Will they be same or diff. species?
DIFFERENT has to be >97%
DNA-DNA hybridization process
- take the DNA from 1 of the organisms
- hit it up to break the H-bonds b/t the 2 strands
- do the same to the other genomes you’re comparing, so you get 2 single strained pieces of DNA from 2 diff cells
- take the opposite strands (i.e. complimentary strands); from 1 cell, & the complementary sequence from the other
- if their as similar as you think, you’ll expect the DNA will hybridize!
DNA-DNA hybridization meaning
putting 2 single stranded pieces; 1 from 1 organism & 1 from another together
- they match up & should be H-bonding b/c the nucleotide should be complimentary
Will all the nucleotides be identical/line up in DNA-DNA hybridization?
just most of them
In the very near future: whole genome sequences?
- it’s cheaper & easier to look at 1 gene rather than whole genome
- but if we can sequence it quickly & inexpensively we’d get most accurate results so would be best
If they have >97% sequence similarity in the 16S rRNA gene, is that 100%/are they identical?
NO!
> 97% sequence similarity in 16S rRNA gene = members of 1 species
- will divide differences that make it not 100% & call it a STRAIN
- allows us to further subdivide those groups of species that are SO similar but not identical into further diversification
- ACCOUNTS for the REMAINING 3%
Opportunist pathogen
waits for the opportunity, can cause disease under right circumstances
ex: e.coli in our body
- does good, but can turn on us
What is interesting of warm bloodied animals like cows & us in terms of e.coli?
Warm blooded animals, like cows also have e.coli like us but it’s NOT the same strain
- we don’t have a lot of homology after that 97% to say that their the same species but not the same strain
- still same species >97% sequence similarity in that 1 gene, but not 100%, so share most diagnostic characteristics but not all
- so if you put a strain in your body from cows you can die even though you have e.coli present in your large intestine
ex: hamburger disease
Describe Classification and Nomenclature
• Microbiologists use Hierarchical classification
- Groups of organisms are placed in successively larger groups
- In practice: Species, genus and phylum are commonly used
ex: Genus of Bacteria
–> species
–> species
–> species
depending on amount of genetic diversity that exits within that group
Describe the binomial species names
Escherichia coli
- Genus (capitalized)
- Specific epithet (not capitalized)
- Strains can be identified by symbols after the species name
- ex. E. coli K12
What are the rules for classification & nomenclature?
Rules
- Names are latinized
- Italicized or underlined
- Genus capitalized, epithet is not
- Genus name may be abbreviated the second time it’s used: E.coli
- Trivial names can be used, but do not follow these rules
What should the strain look like in terms of classification & nomenclature?
ex: 0157:H7 (strain of hamburger disease)
- in NORMAL font
- written after the binomial species names
- not italicized or underlined
What did Robert Hooke discover about microorganisms?
The first to describe microbes
1st guy
How did Robert Hooke become the first to describe microbes?
- Used a compound microscope – uses 2 lenses to magnify the image
- Allowed magnification up to 30x
What did Robert Hooke use the compound microscope to observe?
• Cells in cork (from a TREE - plant cell (euk); bigger than prok. so size wasn’t an issue as it was big so able to see with the 30x magnification)
• Bread mold filaments – 1st microbe
- fungi
• Beginning of cell theory–all living things are composed of cells
What is cell theory?
if it’s alive, cell matter will be the composition
- bacteria, archaea, & all of the diff. members of the domain Euk.
What about the 1st discovery of viruses?
1935 - had diseased tobacco plants, but when they looked under the LIGHT microscope there was no microorganism - so people were not sure why they had a disease
- in 1945, 1st ELECTRON microscope was developed (uses a beam of electrons to provide better magn. & resolution)
- 1st virus was identified - when using e- microscope b/c viruses are smaller than living cells so it wasn’t able to be seen with light microscope
= TMV - tobacco mosaic virus
What did Antoni van Leeuwenhoek discover in terms of microorganisms?
- Built microscopes that magnified specimen by 50-300x
- Observed single celled microorganisms (that were too small for Hooke to observe)– called them “animalcules”
- First discovery of bacteria
(2nd guy)
What did Louis Pasteur discover?
• Studied wine and beer production
1) Yeasts convert sugar to alcohol in the absence of oxygen
- FERMENTATION – “La vie sans air” (life without air)
(lower energy yield)
2) Bacteria can sour wine by converting alcohols to acid
- change taste
- starting with the alcohol as an organic molecule or sugar can be switched to an acid structure which changes the functional group to carboxylic acid which will protonate (cause DECREASE in pH)
3) Developed a method of gentle heating to kill unwanted bacteria – PASTEURIZATION
(3rd guy)
Fermentation
Yeasts convert sugar to alcohol in the absence of oxygen
“La vie sans air” (life without air)
Pasteurization
a method of gentle heating to kill unwanted bacteria
- lower the count in order to protect against disease
Describe pasteurization in terms of a carton of milk
- take a carton of milk & subject it to a mild (NOT destroying properties of milk that you appreciate) heat treatment that will decrease the microbial count present in the milk
- before opening milk for 1st time, its filled with bacteria, but b/c its pasteurized the #’s are much lower, the disease causing bacteria has been killed & you store it at refrigeration temps. which 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?
- see curdling - clumps of bacteria
- b/c heat will kill them & destroy their structure so they aggregate together early on
- & all properties will be ruined b/c of microbial growth that took place inside
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
- Led to the development of methods for controlling the growth of microorganisms (ASEPTIC technique)
What did Louis Pasteur’s spontaneous generation experiment lead to?
• Led to the development of methods for controlling the growth of microorganisms (aseptic technique)
Describe in detail, Louis Pasteur’s spontaneous generation experiment
- Nonsterile liquid poured into flask
- poured nutrient both into flask (has all things needed for most organsims to grow, ex: c source, n source, iron etc.)
- contaminants will be there, bacteria end up @ bottom
- Neck of flask drawn out in flame (steam forced out open end)
- so that things can’t drop into flask
- anything that drops in the flask here will settle @ the bottom, therefore controlling the entry of any new microorganisms that aren’t already there (controlled exp.) - Liquid sterilized by extensive heating
- heating the broth = kill all of those things deposited in there early on
- therefore, setting it to a point of 0
4.
a. LONG time
- dust & microorganisms trapped in bend
- liquid cooled slowly
- left a long time
- liquid remains sterile indefinitely! So, nothing in there that provided repro. capacity for a new cell to come about
b. SHORT time
- flask tipped such that microorganism-laden dust contacts sterile liquid
- he took flask that remained sterile (clear, doesn’t stink, no indication of something growing/spoilage) & turned it on its side, the nutrient broth came down into the troph that was in there
- left a short time
- turned it right side up, introduced bugs that were settled in low point of neck into broth
- liquid putrefies! (spoilage, stench, change in colour, cloudy) indicating that the nutrient medium could support growth it’s just that there was initially no organisms in there
- disproves spon. generation
Aseptic technique
where things happen in such conditions where you try to minimize contamination
- used to avoid contamination & likelyhood of unwanted growth
ex: in OR
- gloved/gowned
- proper ventilation
What did Louis Pasteur want to know?
if spontaneous growth was possible
- if he leaves it knowing there is nothing living present inside the broth, will new microorganisms be able to spon. come about without the nutrient broth, knowing that he feed them everything they will need to be able to increase their cell count - NO!
What did Robert Koch study?
• Studied anthrax – responsible for epidemics in livestock (having a lot of livestock living in close quarters will get anthrax epidemic - become infected & spread it to other organisms living like that)
• Established a set of criteria for relating a specific microbe to a disease
- Koch’s postulates
(4th guy)
How did Robert Koch carry out his experiment?
He isolated bacteria from the carcass of a diseased animal (animals that died of anthrax)– Bacillus anthracis (name of organism that’s going to make the anthrax toxin)
- Injected healthy animals with the bacterium
- Animals became ill with anthrax (supporting hypothesis - that that bug will be responsible for that disease)
- Re-isolated B. anthracis from the test subjects (animals he had infected) and showed that it was identical (organism that was present in these animals, is identical to the one initially isolated from the carcasses)
• Established a set of criteria for relating a specific microbe to a disease
- Koch’s postulates
Koch’s postulates
Robert Koch, established a 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 & absent from healthy animals
- The suspected pathogen must be grown in pure culture
- Cells from a pure culture of the suspected pathogen must cause disease in a healthy animal
- The suspected pathogen must be reisolated & shown to be the same as the original
What are the experimental aspects of Koch’s Postulates?
- Observe blood/tissue under the microscope
- Streak agar plate with sample from either a diseased or a healthy animal
- Inoculate healthy animal with cells of suspected pathogen
- Remove blood or tissue sample & observe by microscopy
- Laboratory culture
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?
could be something else, b/c of the 25 people who might have something else but looks the same so there has to be more so postulates help to address that
What did Robert Koch realize?
Realized that solid media provided a simple way to obtain pure cultures
Describe the solid media
• Broth medium solidified with agar
- Polysaccharide derived from marine algae
- Melts at ~ 97°C and polymerizes (solidifies) at ~ 43°C
- Cannot be degraded by most microorganisms
- Typical PETRI PLATE = nutrient broth medium + 1.5% agar
Typical Petri plate =
nutrient broth medium + 1.5% agar
Solid growth medium vs. liquid growth medium
solid - can pick at it to make a pure culture
liquid (no agar!)- can’t pick at it to make a pure culture
What does agar add?
powder (adds solidifying agent) to liquid medium/broth
- pour & let cool
- once it comes down to ~43 degrees celsius, agar becomes solid & you get a solid support (solidified @ RT)
- provides solid growth medium that allows you ability to create a pure culture
Why is agar used?
b/c microorganisms do NOT have enzymes that allow them to cut the polysaccharide to use it as a source of carbon or a source of ATP
- if no enzymes to cut it, the organism is NOT able to degrade the structure & it remains a solid support underneath the organism
- if the bacteria could secrete enzymes, the solid agar that they sit on top of would be gone, b/c they’d eat right through it
Is it true to describe the broth medium as liquid growth medium?
NO - b/c agar makes it solid
What happens if there is no agar?
it’ll be a liquid medium/broth, b/c nothing to solidify it & you would not be able to use it for pure culture
What is the petri plate made of?
peptone - protein digest that comes from AA’s for growth
beef extract - best way to encourage growth of bacteria - b/c of phospholipids, excellent source of energy, AA’s from protein, nucleotides from DNA/RNA, iron, etc.
NaCl - keep all gradient necessary for transport across the membrane
Agar - solidifying agent
What is the streak plate technique?
- take petri dish that has agar (such as blood (to enrich growth for organisms or to test for certain properties, chocolate etc.) & stick the loop into the bunsen burner to heat up (sterilize & then cool to avoid killing organisms)
- then grab organism with the loop & use another petri dish to spread it on there in 1 location (highly concentrated)
- to dilute it, you flame loop again & set microbial count to 0 again & let it cool & then streak it (diluting out the #’s - becoming a smaller #)
- do it again & again & then you see counts very low compared to before
- will see growth the next day
• 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
What is a colony?
a mass of cells that (ideally) arose from one single cell
• Can be used to create a pure culture
What is the result after streaking/after streak plate technique?
IDENTICAL POPULATION OF CELLS
- same strain (if no genetic mutation)
- b/c they are consuming the nutrients in the growth medium (1 cell –> 2 –> 4 –> 8 –> 16 etc.) at optimal temp (37 degrees celsius)
What is a potential source of error? Is there a way to guarantee if just 1 cell fell into this spot to that colony?
- maybe 2 cells fell really close together
- & each divide into 1 –> 2 –> 4 –> 8 etc.
therefore, colony isn’t from 1 cell in the OG sample that was spread, but could be from 2 (or more)
What is the spread plate technique?
- 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
- Sample is pipetted onto surface of agar plate (0.1 ml or less)
- Sample is spread evenly over surface of agar using sterile glass spreader (that was put into alcohol to kill any contaminating organisms)
- Incubation - for 37 degrees celsius overnight
- Typical spread-plate results - every 1 organism that fell will divide by binary fission to form a colony so you get visible colonies you can count (therefore, 1 cell that fell from the pipette creates 1 colony here)
What happens if you let it in the incubator at 37 degrees celsius for 4 days, for the spread plate tech?
- it will eat all the nutrients - & see an overgrowth (colonies all grown into each other b/c of the excessive binary fission)
- eventually will run out of nutrients & starve (possibly die)
What is the pour plate technique?
- Or can be mixed with molten agar (~ 45°C)
- Colonies form embedded inside the plate
- Use empty sterile petri dish
- Volume of sample
- Pour nutrient broth with agar
- Swirl
- Leave to cool
Since you boil it to form nutrient broth with agar. What will be the problem & boiling temp if you poured it in?
you’d kill all of the organism
- pour when it is cool enough, but still hot enough that its liquid
until ~43 degrees celsius (not more b/c it will harden!)
Where will the organism grow on the spread plate vs pour plate technique?
Spread plate - colonies were present ONLY on top
Pour plate - organism will be at top, middle, & bottom of plate (all THROUGHOUT growth medium)
- multi-dimensional separation!
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 (assume every colony counted began from 1 cell)
- cfu = colony forming unit
The standard plate count procedure
diluting sample
- Pour 1 ml sample into 9 ml broth (fresh, sterile, nutrient broth - no agar!)
- Plate 1 ml samples (spread using spread plate tech), petri dish contains nutritious agar
- Incubation
- If it is a countable plate (30-300 colonies) you can plate count x dilution factor to get cells per ml of OG sample
constantly changing b/c they’re growing, but goal is to control so they don’t
Every 1 cell placed on the plat will dev. into a colony over time, how?
uses nutrients in growth medium to grow @ its own rate
Cooler the temp…
slower they’ll grow
Would 40 colonies be countable? What would you do to the 40 to see what cfu of OG sample you have? What do you hope for the value?
Yes - it’s b/t 30-300
40 x 10 000 (specific dilution factor) = 40 000 cfu/ml
should be the same value to others b/c you corrected for dilution & they all came from 1 ml of OG sample
- to get accurate rep. of thre true # of cells - take average b/c both countable & correct for human error
Describe 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.
Is the bacteria we count living, dead or both?
LIVING - b/c it grew
- VIABLE COUNT
