types of germicides v disinfection level
1. alcohol: intermediate level disinfection
2. aldehydes (glutaraldehyde/formaldehyde):
                        intermediate to high level                                            depending on concentration
3. phenols: intermediate level
4. halogens (chlorine/iodine): ineffective                                       against endospores
​4. peroxygens: high level at high concentrations
5. detergents (SDS and ammonia): low level 

and now we're back to required information below...

summary

                                                                 review question:
Shigella dysenteriae (gram (-) bacteria) requires one of the following solutions to be used as a disinfectant:
1) chlorine for at least 30 seconds.
2) iodine for at least one minute.
3) ammonia for at least one minute

Would you consider this bacteria to require low, medium or high level of disinfection.
​Why?

review question:
what is the difference between microbiostatics and microbicidals?

many heterotrophic bacteria that live in the soil can
 "fix" large amounts of nitrogen converting nitrogen in the     atmosphere to non-gaseous nitrogen such as ammonia that can   be used then as a source for energy. 

 spore formation

pressure

culturing anaerobic bacteria

defined/complex/differential/selective media

review question:
describe the categories of bacteria in reference to oxygen requirements.

 
  Nt = number of cells at a certain time point

  No = initial number of cells

   n = number of generations

review question:
what is nitrogen fixation and why is it imnportant?

C6H12O6

review question:
list the 4 physical methods of
microbial control and give an example of each.

microbial growth in a laboratory is very different from microbial growth in real life.
 in a lab setting, ​microbes are:
*grown in nutrient-rich media for rapid growth
*grown in precise temperatures for optimal growth
*grown in pure cultures (only one type of microbe)
  without competition
*grown with favorable pH and O2 levels

So, in a lab, there's often rapid exponential growth while
in nature, growth is slower and inconsistent.  

review question:
autotrophic bacteria fall into two categories.

list them and explain how they are different.

optimum growth rate vs pH

 isolating microbes permits you to study the characteristics of specific species and
is necessary for identifying bacteria that cause diseases

you can isolate via:
 streaking a mixed culture across a solid agar plate to isolate individual colonies.
each colony is a single microbial species. 

plating a diluted samples of a mixed culture across the surface of an agar plate, allowing individual colonies to develop. 

pour plating microbes mixed with molten agar and pouring into a petri dish to create a lawn of growth. After incubation, individual colonies can be isolated by picking them from the plate.

​

serial dilution reduces bacterial load.
a sample is diluted repeatedly until such point

that individual cells can be cultured on a solid agar plate and colonies selected.

​

review question:
what are three imperatives when collecting bacterial samples?

stationary phase:
​exponential growth cannot continue indefinitely due to dwindling food and increasing wastes leading to stationary phase

review question:
​
a population of
M. tuberculosis
will double every 14 hours. 
If you start with 50 bacteria in a closed batch system, how many bacteria will you have after 56 hours?

sporulation (spore formation) allows bacteria to survive harsh conditions. 

 
  g = generation time (minutes or hours)
  t = time of exponential growth
  n = number of generations
​        

 chemoautotrophic bacteria
 while photoautotrophs use light as their primary energy source,    chemoautotrophs obtain energy from the oxidation of inorganic or organic molecules. 

 binary fission

     salt loving bacteria??
yup, some bacteria thrive in very salty conditions. salty = high NaCl

they're called halophiles

optimum growth rate vs oxygen concentration

 most bacteria are neutralophiles which thrive
at neutral pH levels.
ex: Escherichia coli
Staphylococcus
 Salmonella  

 clinical equipment is categorized as:
1. critical requiring sterilization
2. semi-critical requiring high level disinfection
3. non-critical satisfied with low-level disinfection 

critical equipment contact tissue or the bloodstream
                                   ex: surgical instruments and implants. 
semi-critical equipment contact mucous membranes or                                             broken skin
                                   ex: endoscopes, endotracheal tubes
non-critical equipment contact intact skin
                                   ex:  blood pressure cuffs, bedrails

collection methods

review question:
bacteria that are pathogenic to humans are usually mesophiles. Why?

         germicide activity
1. high-level disinfection
     kills most pathogens including spores
2. intermediate-level disinfection
     kills vegetative microorganisms, some           viruses, and fungi
3. low-level disinfection
     kills only vegetative bacteria and some         enveloped viruses

 try this problem.
submit your answer in writing in class Monday 3/10.

please show all of your work.

​Salmonella enterica has a  generation time
of 30 minutes. If you start with
10 bacteria, how many will you have in 3 hours?

temperature

                 classes of germicides
1. antiseptics kill microbes on living tissue
2. disinfectants kill microbes on inanimate objects  

essential nutrients are molecules required to build new cells and are found in the environment of the microbe.
they may be micronutrients or macronutrients.  

the time it takes for a bacterial population to double in size is called the
generational time

generation time  
​is determined during the exponential growth phase
(log phase)

bacterial growth

                              how to decontaminate for:
mycobacteria (causes TB and leprosy):
disinfection: heat sterilization via autoclaving
                        hydrogen peroxide/glutaraldehyde

endospores (anthrax, C.diff):
disinfection: autoclave, glutaraldehyde

viruses: 
disinfection: heat sterilization, glutaraldehyde or chlorine
                        detergents

protozoa:
disinfecion: boiling water can kill many protozoan.
                      cysts require iodine compounds or exposure to                          UV light

prions:
disinfection: extended autoclaving 134°C for 18 minutes or                            132°C for 1 hour

nitrogen fixation

     
                   step one: calculate n
   (how many generations will there be in 2 hours)
                         we are solving for n
                    n =  number of generations
 
 1.  first we have to figure out how many minutes are        in two hours.
      there are 60 minutes in one hour
      so "t"  would be = 2 hours x 60 minutes per hour
      "t" = 120 minutes 

 2. second we set up the equation    n = t/g
      n = 120 minutes time period/
                                       20 minutes generation  time
      n = 6 generations in 120 minutes

death phase:
bacteria run out of nutrients and die. 

some bacteria use O2 during a process of cellular respiration.






​

this process assembles a molecule called ATP.
ATP is used by the cell to store its energy.
​ the cell uses the energy stored in food to assemble the ATP. 
​

energy in food is stored in the covalent bonds of the atoms that make up food.

the cell processes food as glucose.
​Glucose is C6H12O6.


​






​
the release of energy (exergonic reaction) when glucose is broken down is used to bond together atoms to form a different molecule called ATP. 

Once the energy is released from glucose breaking apart,
the atoms of C, H and O from the C6H12O6 have leave the cell.
these atoms are now considered waste products.
the C and the O from the C6H12O6 bond together to form CO2 which is carbon dioxide. the CO2 is released from the cell as waste gas. 
well, what to do with the H?
here's where the oxygen that the cell "breathes" in is used.
 this oxygen bonds to the Hydrogen left over from the glucose.

Oxygen and Hydrogen bond to form water H2O.
the H2O leaves the cell as waste. 

so oxygen is taken in by cells so it can bond to the leftover hydrogen from your food

which forms which is then released as waste. its true!  

if oxygen is not present, cells may rely on a different process to build ATP called
 fermentation. whole other story...

xtra credit question:
explain the function of
oxygen during cellular respiration

 generation time
​can be visually determined by 
plotting the log phase cell number vs time.
 The slope of the line is  predictable. 

two formulas used

why do we grown microbes in a lab?
*to learn about their biology
*to identify pathogens
*to develop treatments
*to identify possible applications such as in biotech

 autotrophic bacteria
  use a process called
carbon fixation
to convert carbon dioxide into organic compounds
they can then use as a food source

decontamination:

sterilization vs disinfection

each of these bonds was made using energy and that energy is stored in that bond. 

 E. coli doubles in 20 minutes under ideal conditions but if nutrients are sparse, that number is significantly longer. 

review question:
what is a microbial growth factor?

​Give two examples.

review question:
describe one adaptation used by bacteria to accommodate the extremely high or low  concentration of H+ of their environment. 

  we use generation time in to predict how bacteria will grow in different environments. 

liquid/solid/semi-solid

other bacteria can withstand and even thrive in acidic or alkaline conditions.
one acidophiles is Picrophilus.
it thrives in crazy acidic environments
like volcanic lakes exhibiting a pH of 0.06. 

one alkaliphiles is Alkalibacterium tumefaciens
it thrives in environments with pH levels > 10

exponential phase (log/logarithmic phase):
doubling will continue at a constant rate =
the # of number of cells doubles with each time interval. plotting the cell number vs time produces a straight line and the slope = growth rate of the bacteria.
the slope of this line depends on growth conditions.

why?
food safety and healthcare requires estimates of  the number of microbes in a sample.

how?
1. Colony Forming Unit (CFU) Plate Count
you can estimate bacterial cells in a sample by diluting the sample, plating onto agar plates, and counting the resulting colonies after incubation. 
then you can calculate the number of bacteria (CFU) per unit volume or mass of the original sample. counting microbes

 microbial carbon fixation removes CO2 from the atmosphere and incorporates   it   into our planet's biomass. as such , they are super important in our global ecosystem.
 microbes are used in wastewater treatment to remove pollutants and convert CO2 into biomass. the potential of using bacteria for CO2 fixation and conversion into   biofuels, bioplastics, and other products is being researched. 

autotrophic vs heterotrophic bacteria

molecules that a bacterium can't make on its own and must get from outside the cell are called growth factors. 
examples: vitamins, amino acids, nucleotides

optimum growth rate vs salinity

xtra credit question:
read this article
https://pmc.ncbi.nlm.nih.gov/articles/PMC7024382/
what is described as an application of halophilic bacteria in medicine?

lag phase:
bacteria adapt to growth conditions as they mature

review question:
describe one adaptation used by bacteria to accommodate the extremely high or low salinity 
of their environment. 

ex: C. botulinum 
​and
C. perfringens

 E. coli can double in 20 minutes
 M. tuberculosis doubles in 14 hours. 

mesopohiles

some bacterial species utilize oxygen differently than others.
obligate aerobes require O2 to survive
obligate anaerobes can't tolerate O2
facultative bacteria can grow with or without O2
bacteria needing very low O2 levels are called microaerophiles

review question:
bacterial growth curves rely on the use of a closed batch system during culturing. what is that?

this pic shows the covalent  bonds of glucose

review question:
explain why you would serial dilute a culture before plating.

aerobic vs anaerobic bacteria

     adaptations to permit growth in extreme pH:
*cell membrane modifications
*proton pumps which pump H+ out of the cell to maintain a               neutral pH
*specialized proteins that can do not denature
        (maintain their structure) under extreme pH
* pH sensing mechanisms can detect changes in outside pH               and respond to maintain homeostasis. 


 acidophiles (low pH) exhibit highly charged proteins and        membranes resistant to H+
 alkaliphiles (high pH) exhibit negatively charged cell walls and use sodium pumps to help maintain pH 

turbidity
turbidity measurement usually requires
 spectrophotometer to indirectly estimate cell density. you measuring the amount of light transmitted through a sample.
 higher turbidity correlates to a higher cell count. 

        when collecting bacterial samples:
sterile technique!
use sterile containers and equipment
ensure sample is transported to the lab quickly and under appropriate conditions

wound culture:
swab, syringe, biopsy clean wound tissue surface culture:
swab across surface then streak agar plate water sample:
collect the sample in a sterile container. 

use sterilize containers, swabs, etc.
collect samples in sterile containers then
transport samples to the laboratory quickly
to maintain sample integrity. 
document the sample's path, including the date, time, and collector's name if this data is needed.

review question:
do you think bacteria that are pathogenic to humans would be extreme alkaliphiles? explain your answer.

review question:
describe three methods of bacterial replication

radiation

ex: cyanobacteria

isolating microbes

pls reference our
​ discussion from lab

cyanobacteria have a phenomenal carbon-absorption rate, credited
for removing 25% of our plane'ts atmospheric CO2!

culturing microbes

optimum growth rate vs temperature

germicides ​
kill or inactivate microbes
1. antiseptics ex: hand sanitizers and iodine
2. disinfectants ex: bleach, hydrogen peroxide, glutaraldehyde. 

ex: streptococcus, bacillus, E coli

Halophiles adapt to osmotic stress by storing loads of
organic solutes in their cytoplasm.
this is called an 
​osmolyte strategy.
which helps balance the high salt concentration of their
environment.

Environmental factors influence how well bacterial grow including pH, temperature, water activity, macro and micro nutrients and O2

review question:
explain the differences between high, intermediate and low level disinfection.

review question:
anaerobic bacteria must be cultured in an environment devoid of oxygen because...

microbiostatics inhibit the growth or multiplication of microorganisms without necessarily killing them.
they slow down or prevent the reproduction and growth of microorganisms.
ex: refrigeration, certain preservatives
vs
microbicidals which kill microbes
ex: disinfectants, antiseptics, and some antibiotics

review question:
what is the difference between sterilization and disinfection?

​ generation times
differ based on species and environmental conditions. 

 how do bacteria replicate?

review question:
what is meant by a bacterial species generation time?

water

FYI heat treatment terms: 
decimal reduction time (D-value) is the time required to reduce a microbial population by 90% under specific conditions re temperature or sterilant

thermal death point (TDP) is the lowest temp at which all microbes are killed within a certain time frame. this is used for assessing the effectiveness of heat sterilization and food safety

thermal death time (TDT) is the shortest time needed to kill all microbes at a specific temperature. Used to determine effective heat treatment protocols

   manipulating
   the equation:
       n = t/g

review question:
what are essential nutrients. list the two categories of essential nutrients and give an example of each.

decontamination

below info explains what oxygen is used for during metabolic reactions. it is not required information

review question:
explain the four stages of a a bacterial growth curve.

decontamination:
includes cleaning, disinfection, and sterilization aimed to make an
an area, device or material safe to handle.

review question:
how would you relate the turbidity of a culture to its bacterial load?

calculating
​ generation time

review question:
compare differential v selective media.

review question:
a population of E coli is being grown in a closed batch system. E. coli has a generation time of 20 minutes.
you start with 10 cells.
​how many cells will you have after 3 days?

 macronutrients include carbon, hydrogen, oxygen, nitrogen, phosphorous, 
sulfur, potassium, calcium and magnesium
​
 micronutrients include iron, zinc and copper

review question:
how do most heterotrophic bacteria get their energy?

review question:
what are the two classes of germicides?

 some halophiles also rely on a
 salt in strategy
    stockpiling inorganic ions like KCl in their
cytoplasm to balance
 the salinity in their environment. 

  effect of environmental influences on bacterial growth

review question:
describe the three categories of clinical equipment as they relate to disinfection/sterilization.

nutrients and growth factors 

physical methods of microbial control include:
temperature
pressure

moisture

radiation

chapter 7:
microbial growth and decontamination

plants cannot directly use the nitrogen in the air. they rely on bacteria to convert it to forms that plants can absorb and use for building proteins and nucleic acids.

many bacteria have a symbiotic relationship with plants, providing them with fixed nitrogen in exchange for carbohydrates. 

nitrogen fixation reduces the need for farmers to apply for fertilizers,
can improve the quality of the soil and improve crop yields in ways that are environmentally sound. 

counting microbes

review question:
why is it useful to know the generation time of a bacterial species?

 budding

 optimum: growth rates are the highest at the optimum temperature/pH levels
  minimum: the lowest temperature/pH at which the organism can survive and
                       replicate is its minimum growth temperature/pH.
  maximum: the highest temperature and pH at which growth can occur is its
                       maximum growth temperature and pH.

example:
E. coli has a generation time of 20 minutes.
​If you start with one cell, how many cells will you have after 2 hours?

review question:
gram positive bacteria are easier to combat via disinfection practices than gram negative bacteria. why do you think that is?

bacterial growth measurements rely on a 
"closed pure batch system"
where all the required ingredients for growth are added to the culture at the start of the process. nothing else is added or removed during  the reaction. this type of system produces a predictable growth curve exhibiting four distinct phases:
lag, exponential, stationary, and death

 step two: calculate Nt
  (number of cells present after
    6 generations if you start with one E coli cell) 

Nt = N0 x 2^6

Nt = 1 x 64

​ 64 cells will be present after 2 hours 

generation time
also differ based on factors such as temperature, moisture and the availability of nutrients 

meso = middle