Micro Ch.6 Microbial Growth

Acidophiles

Acidophiles are bacteria resistant to low pH such as the some Chemoautotrophs which are found in drainage of coal mines and oxidize sulfur to sulfuric acids, and can survive the pH of 1. Bacteria often produce acids that may interfere with their growth and the solutions are buffered to prevent pH change.

Metabolic activity

Another indirect way to estimate bacterial growth is to measure metabolic activities such as measuring acids, or co2 to determine bacterial growth.

Extreme halophiles

Are adapted to high concentration of salt and require high salt. * salt-tolerant prokaryotes * they can maintain optimal osmotic pressure without suffering from plasmolysis

What are Obligate anaerobes?

Are bacteria that unable to use oxygen and are harmed by oxygen. The genus clostridium which contains species that case tetanus and botulism are the most familiar examples. Several forms of toxic oxygen exist as follow: - 1-single oxygen (1o2 -, which is highly reactive, 2-superoxide Radicals (o2- ) formed during normal respiration very toxic, Organisms living in the atmosphere must possess superoxide Dismutase (sod) to neutralize them. Anerobes, faculatative Anaerobs and aertolerant must produce sod that converts Superoxide radical to hydrogen peroxide (h2o2). 3- hydrogen Peroxide produced contains peroxide anions (o2 2-) which is toxic, Hydrogen peroxide is broken down with catalase which produces H2o+o2, or by peroxidase that produces h2o. 4- hydroxyl radical, is Most reactive and produced in cytoplasm by radiation, but they are Transient.

Microaerophiles

Are those that require oxygen less than the amount present on the atmosphere. They grow on a very limited range of Oxygen

Turbidity method

Bacteria are multiplying become more turbid as the bacterial numbers increase. This could be measured by a Spectrophotometer determining turbidity of the bacterial culture. As the bacteria grows, less light is transmitted through the system which is determined as percentage transmission, registered as logarithmic expression called As absorbance (also called optical density) is used to assess growth. If the absorbance matches plate count, this could be used to determine the bacterial numbers.

How does bacteria reproduce?

Bacteria normally reproduce by binary fission which components are doubled and the bacteria is divided into two bacteria of equal size.

Obligate Aerobes

Bacteria that require oxygen to live

More special culture techniques..

Candles are burned in certain jars to produce carbon dioxide. Certain bacteria such as ricketssia and chlamydia require tissue for growth. Some organisms that are highly infectious are handled under extraordinary condition and systems of containment called biosafety levels 1- 4 (bsl1-4). There are few bsl-4 labs being used in areas called hot zone.

Aerotolerant

Cannot use oxygen, but they are tolerant, most ferment carbohydrates to lactic acid and as lactic acid accumulates, oxygen level is reduced in the environment which provide a more suitable environment for bacteria to grow. Examples are lactobacillus used to make pickles and cheese.

What is log phase?

Cells begin to divide and increase their numbers exponentially. Because generation time is constant, log phase is straight line. Because In log phase, cells are most metabolically active, it is preferred for industrial purposes, for example when products are needed to be produced efficiently.

What types of bacteria are grown in armadillo under special culture techniques?

Certain bacteria requires specific environment, Mycobacterium leprea, the Leprosy Bacillus, is grown in armadillo because of its low body temperature.

Pour plate method

Cfus are separated from one another using a series of dillutions. colonies form both at and below the surface of the medium Specifics of Pour plate: In pour plate, 0.1 or 1.0 ml of a solution containing diluted bacteria are mixed in to a nutrient agar medium in liquid phase and poured on a plate. When agar is solidified, plate is incubated and colonies are counted after 24 hrs. This method has some draw backs since some bacteria are heat sensitive and melted nutrient agar has a temperature of 40c or higher.

What are the different types of media used for different types of bacteria?

Chemically defined media complex media anaerobic growth media Selective and differential media

Faculatative halophiles

Could live up to 2% salt, a concentration that only a few organisms can tolerate. Some facultative aerobes can tolerate up to 15%. do not require high salt concentrations, but can tolerate them

What is selective media designed to do?

Designed to suppress growth of unwanted bacteria and support growth of the desired microorganisms. For example media with bismuth sulfite agar is used to isolate Sallmonella typhi, while preventing gram positive bacteria and most gram negative bacteria.

Binary fission is not the only way bacteria reproduce. How else can bacteria reproduce?

In some cases, bacterial reproduction is done through budding which is formation of a small initial growth, until its size approaches the adult and then they separate.

what are the trace elements?

Iron, magnesium, molybdium, and zinc function as cofactors and are found in soil.

What is culture media?

Is a nutrient material prepared for bacterial growth, microbes that are introduced in to the culture medium are called inoculum, and the microbes that grow and multiply is called a culture.

What is Enrichment culture?

Is a selective media that allows surviving of organisms that can metabolize specific chemicals. **The surviving organisms are subsequently transferred to a media containing the specific ingredients, and the original media is gradually diluted.

Anaerobic Growth Media

Is for cultivation of anaerobic bacteria and since anaerobic bacteria are killed by oxygen, reducing media should be used which contains ingredients such as sodium thioglycolate that binds to oxygen and depletes oxygen. The plates are placed in sealed boxes, and the oxygen is removed chemically from the box with the aid of hydrogen and co2.

What is bacterial growth?

Is referred to the increase in bacterial numbers.

Chemical requirement: Carbon

Is required for microbial growth, half dry weight of bacterial cell is carbon, chemoheteroptrophs obtain their Carbon from organic compounds, chemoautrophs and Photoautotrphs obtain their carbon from atmospheric co2.

What is Biofilm?

Is the layer and the community that bacteria develop. It is produced by microorganisms which is a layer composed of polysaccharides and peptides. Biofilms are 1000 fold more resistant to antimicrobial reagents. 70% of bacterial infections are due to biofilms according to cdc. Most infections in health care facilities are due to biofilm

Direct measurement of microbial growth: Plate count

Is the most frequently method of measuring live bacterial population. An important advantage is that it measures viable cells. The disadvantage is that it takes 24 hrs to get the results, which can be a problem in some applications such as quality control of milk not being possible to hold a lot that long. Plate Count assumes each live cell forms a colony, since it is not always true and colonies could form from a string of bacteria, plate counts are often reported as colony-forming units (cfu).

What is the death phase?

Is when number of dying cells exceeds numbers of newly formed cells which is also called logarithmic decline phase. This phase continues until the population is diminished to a tiny fraction

What is agar?

It is a semi solid gelatin like polysaccharide with added components necessary for bacterial growth. Agar has important properties, it liquefies at 100 deg Celsius and solidifies around 40 deg Celsius. It cannot be digested by most Bacteria. It is placed in containers called a petri Dish

What is the lag growth phase? What occurs?

Lag phase has very few cell numbers and cells do not reproduce immediately in a new median. This periods which cells have little or no dividing is called lag phase.

What is used to show population change?

Logarithmic numbers are used instead of the normal arithmetic numbers. For example the population of 1,000,000,000 could be shown as logarithm of the number which is 9.

What is differential media designed to do?

Makes it easier to distinguish colonies of different bacteria growing on the same plate. For Example, blood agar used to identify streptococcus Pyrogenes causing strep throat. Colonies from this bacteria form a cleared area around the colonies by lysing red blood cells. Sometimes both selective media and differential media are place together in with agar.

What are capnophiles and what do they require?

Many organisms require low oxygen and high Co2 such as bacteria that live in the intestine. Bacteria that require high co2.

Mesophiles

Most common bacteria are mesophiles. Most pathogenic bacteria have optimal growth temp higher than 37 deg Celsius. Most disease causing bacteria and those responsible for diseases are mesophiles .temperature microorganisms live/prefer: moderate * thrive at medium temperature * range: 10° - 45°C * pathogens thrive in the human body * they are mesophiles

Complex media

Most heterotrophs bacteria and Fungi are grown in complex media composed of yeast Extract, meat or plants, or digested proteins. Their composition varies from batch to batch. Peptides are provided by proteins digested and vitamins are provided by meat extract or yeast. Yeast extract is rich in vitamin B and used in a liquid form called Nutrient broth, when added to agar, it is called Nutrient agar.

What is the temperature requirement for microbial growth?

Most organisms grow well at the same temperature as humans favor. However certain bacteria could grow in EXTREME environments that hinders survival of most eukaryotes.

What is MPN?

Most probable numbers. Bacteria are inoculated in 5 test tubes with 10 ml, 1 ml or 0.1 ml. Bacteria will grow in some of these tubes and not others. A mpn table is used to estimate numbers of cells in the solution with 95% certainty. (table 6.19 b)

Chemical requirement: Nitrogen

Nitrogen is used for DNA, RNA and protein. They obtain nitrogen from decomposition protein, from nitrate ion and some bacteria obtain nitrogen from the atmosphere through a process called nitrogen fixation. Bacteria capable of nitrogen fixation live in a symbiosis conditions with Legumes plants in their roots. This includes, alpha-alpha, beans and Soybean, clover and pea.

Dry weight method

One way to measure growth of filamentous Organisms is their dry weight, since some are spore forming And may not form colonies which makes using plate count less accurate

Phosphorous

Phosphorous required for synthesis of nucleic acids and ATP and Phospholipids, its source comes from phosphate ions.

Microorganisms are classified into 3 groups depending on there preferred range of temperature. What are the 3 groups and their preferred temperature range?

Psychrophiles: cold environment Mesophiles: bacteria that lives in moderate environment Thermophiles: bacteria that lives in hot environment (They also have a minimum growth temperature, optimal growth temp, and maximum growth temperature)

What does the pH requirement for microbial growth refer to?

Refers to a solution's acidity or alkalinity. Most bacteria grow best in a narrow range 6.5-7.5, and very few grow at the pH below pH 4. Which is the reason food like sauerkraut and cheese are preserved and do not spoil.

How is bacteria preserved?

Refrigeration will prolong bacterial survival. Deep freezing is placing pure bacterial culture quick frozen and kept at -50 to -95c. Lyophilization or freeze drying is when a microbial cell suspension is quickly frozen and kept at -50c to -75c.

Obligate halophiles

Require salt to live, with nearly 30% are found in Dead sea.

Sulfur

Sulfur is found in amino acids used for Protein synthesis and in vitamins such as biotin and thiamin. Sulfur is obtained from h2s (hydrogen sulfate), sulfate ions and sulfur amino acids.

Serial Dilution

THE FDA counts plates between 25-250, some microbiologist count between 30-300. To assure this number is met, the original sample is diluted several times in a process called serial dilution. Serial dilution is carried out by sequential dilutions of 0.1 or 1 ml of a sample 4-5 times in different test tubes. A small volume of the diluted (0.1 or 1.0 ml samples are then applied to a plate and colonies that appear after incubation are counted.

What are the physical requirements for microbial growth?

Temperature, pH, and osmotic pressure

What is done to the culture media?

The media has to be sterile at the beginning and have the components necessary for bacterial growth. This includes water, and optimum pH. When it is desired to grow bacteria on a solid medium, agar is used.

If log phase is allowed to continue, it reaches a point when newly formed cells will equal the dying cells and the cell number will stay at the same level for a while. What phase is this called?

The microbial phase is called Stationary Phase. **Factors that may play a role in creating the stationary phase are depletion of nutrients, change in pH and increase in waste products.

In order to obtain a pure culture what method is used?

The streak plate method is used. 1. A sterile inoculating loop is dipped into a mixed culture that contains more than one microorganism. 2. The loop is streaked on a plate in a specific pattern which results in colonies generated from individual microorganisms.

Define Generation Time

The time required for a cell to double its numbers. **It varies among different organisms. Most bacteria require 1-3 hrs, other require 24 hours. If doubling time is 20 minutes, an organism will have over 1 million cells in 20 generations.

What are the phase of microbial growth?

There are four basic growth phases, lag, log, stationary, death.

Indirect methods of measuring microbial growth

Turbidity, metabolic activities, and dry weight

What is direct microscopic count?

Use a 0.01 ml solution containing bacteria. On a cell counter and use a microscope to count the cells. Advantage is it can be done rapidly, the disadvantage is it can not Tell the dead from live cells and requires large number of cells. Automatic electronic cell counters called Coulter counters are Used to count cells.

Spread plate count

Uses 0.1 or 1.0 ml of a solution containing diluted bacteria and plated and spread by a glass hocky stick and bacterial colonies are counted after 24 hrs.

Filtration method

When bacterial numbers are low such as in lake water or ocean, 100 ml of water is filtered retaining bacteria. The filter is then soaked on a liquid nutrient medium where colonies appear from the bacteria on the filter. This method is applied to detect and enumerate coliform bacteria indicator of fecal contamination.

optimal temperature

* Its for prokaryotic species * optimal temperature for growth * acceptable temperature about 30°C

psychrophiles

* temperature microorganisms live/prefer: cold * optimally grow below 15°C * makes up the largest portion of all prokaryotes on earth * they live at the bottom of the sea * they can maintain optimal osmotic pressure without suffering from plasmolysis Certain organisms can grow at 0 deg Celsius, and have optimal growth at 20-30 deg Celsius , BUT CAN NOT GROW AT 40 deg Celsius WHICH ARE IMPORTANT IN FOOD SPOILAGE called psychotrophs.

What is chemically defined Media?

*Is a media that its exact components are known, which require carbon, Nitrogen, sulfur, and other chemicals called Organic growth factors. *Some bacteria require many organic factors, they are called fastidious. *Which are sometimes used to determine concentration of nutrients such as vitamins. The test used is called microbiological assay.

what is osmotic pressure requirement for microbial growth?

*Microorganisms obtain their nutrients from surrounding waters. Their composition is 80-90% water. High osmotic pressure (hypertonic, high salt) can remove water from the cells causing cytoplasm to collapse, a condition called plasmolysis, causing bacterial cell destruction. High salt or sugar is used which prevent bacterial growth and is used to preserve food, fish, and meat.

Oxygen as a chemical requirement

*Oxygen in its form could be toxic, for most of the earth life, a minute amount of oxygen has been present. *Most living forms at the present time, require oxygen in their metabolism. By combining hydrogen and oxygen, energy is formed and the toxic effects of oxygen are neutralized in the cells. *Organisms can extract more energy from chemicals by utilizing oxygen comparing to those not using oxygen.

Faculatative Anaerobes

*Since water does not dissolve oxygen well, many obligate aerobes have developed capability to grow at low oxygen or in the absence of it. *These organisms could use oxygen when oxygen is present, but can perform fermentation such as yeast and also intestinal E.coli

What are the specific chemical requirements for microbial growth?

carbon, nitrogen, Trace elements, and oxygen

Thermophiles

temperature microorganisms live/prefer: hot * thrive around 50-60°C * lives in compost heaps and hot springs There are also HYPERTHERMOPHILES OR EXTREME THERMOPHILES, REQUIRE 80 deg Celsius and some live 121 deg Celsius in thermal vents deep in the ocean. Hyperthermophiles: * temperature microorganisms live/prefer * But mostly they are Archaea * archaea prefer extreme environments * optimally grow in above 80°C * found in seafloor, hot-water vents