Chapter 6: Microbial Growth Flashcards
Microbial growth
Increase in # of cells of microorganism
Psychrophiles
Psychrophiles optimal temp: for growth 15 degrees celsius
Max growth temp: 20 degrees Celsius
Psychrotrophs
-For growth: 25 degrees celsius
-Growth between 0 and 20-30 degrees celsius (refrigerator 4-8)
-Cause food spoilage
-Ex. Listeria monocytogenes
-Deli meats, unpasteurized dairy products
-Causes sepsis and meningitis in neonates and immunocompromised pt’s
-Multiplies inside phagocytes
Mesophiles
Grow at 25-40 degrees celsius
Optimal temp.: 35-37 degrees celsius (body temp)
Ex. pathogens
Thermophiles
-heat-loving
-produce endospores
-Grow best between 50-60 degrees celsius
-Ex. Clostridium and Bacillus (both gram-(+) rods)
Hyperthermophiles
-heat-loving
-produce endospores
-Grow 80 degrees celsius or higher
“Extreme thermophiles”
Food preservation danger zone
-25 degrees Celsius (room temp)
-rapid growth of bacteria
-some may produce exotoxins➡️ food intoxication when ingested
-common cause of food intoxication: Staphylococcus aureus
-cause vomiting and stomach discomfort in healthy pt’s
Physical requirements for growth: pH
-Most bacteria grow between pH 6.5-7.5
-Molds and yeasts grow at pH 5
-Acidophiles grow at pH <4
Extreme halophiles
-grow in ⬆️[salt]
-30% NaCl solution
-found in seawater
Obligate halophiles
-require salt to grow
-optimal salt concentration: 15-30%
Facultative halophiles
-do not require salt but can grow w/ salt present (2-15% salt can grow)
-Ex. Staphylococcus
Requirements for growth: chemical requirements
-Nitrogen- component of AA’s
-Carbon- synthesis of molecules that make up a living cell
-Sulfur- in some AA’s
-Phosphorus- for ATP; in phospholipids and nucleic acids
-hydrogen- source of e-‘sand component of organic molecules
Chemical requirements for growth: trace elements
-Inorganic mineral elements required in small amounts
-Usually act as enzyme cofactors (metal ions)
-Ex. include iron, copper, zinc, magnesium
Obligate aerobes
-require O2
-ex. Bacillus anthracis
Grows toward top of tube
-optimal amount of O2: 20%
Facultative anaerobes
-Grow w/ or w/o O2 (through anaerobic respiration or fermentation)
-Grows throughout tube (but more concentrated toward the top)
-optimal O2 level: 20%
Obligate anaerobes
-harmed by O2
-ex. Clostridium
-grow at bottom of tube
-Gram-(+)
-endospore -forming
-Rod
-Put in anaerobic jar
-Cause botulism
Aerotolerant anaerobes
-use fermentation to produce ATP
-are not affected by O2
-will grow evenly distributed throughout the tube, meaning it can be found at all levels, from the top to the bottom, as it can tolerate oxygen but doesn’t require it to grow
-ex. Lactobacillus
-rod
-gram-(+)
Microaeophiles
-require low O2
-optimal O2 level: 2-5% O2
-Find area in tube where O2 is 2-5%
-Campylobacter (causes diarrhea and salmonella) and Helicobacter
What will occur if bacterium that normally lives is GI tract is plunged into salty solution?
Plasmolysis (cell losses water and shrinks)
Chemical requirements: organic growth factors
-organic compounds that bacteria need to grow
-Essential compounds that the microbe can not make itself
-Fastidious organisms
-one with complex or specific nutritional needs, making it difficult to grow in a laboratory setting
-Ex. AA’s, vit’s, purines and pyrimidines, NAD+
Biofilms
-Microbial communities form slime of hydrogels
-Clumps of bacteria adhering to a surface- Attached to each other, and the surface is attached to the water
-microbes in biofilms are more resistant to antibiotics than a free-swimming microbes
Characteristics of biofilms
-Bacteria attracted by chemical signaling
-Sheltered/protected from harm factors (antibiotics, disinfectants and host defenses)
-heterogeneity of biofilm community- constantly changing in structure and composition over time
Pseudomonas
-found in wet environments like soil and water
-often involved in biofilms
-Pseudomonas aeruginosa
-survives harsh environments
-common in hospitals and plant surfaces
Culture media
-cultured medium is any material prepared for the growth of bacteria in a lab
-Nutrients prepared for microbial growth
-Sterile: no living microbes- autoclave
-Inoculum: intro of microbes of medium
-Culture: microbes growing in/on culture medium ((s) of (l))
-1) Solid media- nutrients in petri dish containing agar-nutrient
-2) liquid media- nutrient broth in test tube
Criteria for culture media
-Sterility
-Proper nutrients
-Proper pH (7) and incubation temp. (35-37 degrees celsius- in this course to stimulate bacterial growth)
-Proper incubation atmosphere (O2, CO2, etc,)
Agar
-solidifying agent for a culture medium
-gelatinous substance derived from red algae
-Agar is used to create solid growth areas for bacteria in microbiology labs. Agar plates can be selective, meaning they can be used to promote the growth of certain bacteria while inhibiting others
-Complex polysaccharide
-A solidifying agent
-Generally not metabolized by microbes (degrade)
-Liquefies at 100 degrees celsius
-Solidifies at about 40 degrees celsius
Chemically defined media (CDM)
-sterile, liquid growth medium used to culture cells in a laboratory setting
-It’s made up of known chemical components, and is free of animal-derived product
-CDM provides a stable environment for cells to grow in.
-CDM leads to more consistent experimental outcomes.
-CDM allows for better control of performance in cell culture.
Reducing media
-Sodium Thioglycolate chemically rem’s O2
-culturing obligate anaerobes (require O2-free environment to survive)
-contains ingredients that remove oxygen from the medium, making it suitable for anaerobic bacteria.
How does reducing media work?
-Reducing agents, like sodium thioglycolate, are added to the media to chemically combine with dissolved oxygen.
-Boiling the media can also help remove dissolved oxygen
Enriched media
-encourages growth of fastidious organisms (specific nutritional needs that make it difficult to grow in a lab setting) that need organic growth factors
-nutrients in enriched media help ensure the survival and multiplication of these microorganisms
-ex. culture medium with a high salt concentration can be used to grow salt-tolerant bacteria
-used to grow pathogens including:
-Neisseria gonorrhoeae
-Neisseria meningitidis
-Haemophilus influenzae
-made by adding nutrients like blood, serum, or egg yolk to a basic medium.
Trypticase soy agar
-ex Micrococcus letus
-yellow pigment
Chocolate agar
-supplements added to media such as Hb powder, vit B12, AA’s, and NAD
-growth medium used to isolate and identify bacteria
-variant of blood agar that’s made by heating blood cells to lyse them. The heated blood gives the agar a brown color and a “chocolate” appearance.
-ex. Pathogenic Neisseria, Haermophilus
-Grow fastidious organisms that won’t grow on other types of agar
-Test aerotolerance of obligate anaerobes
-grow all types of bacteria, including Gram-positive and Gram-negative organisms.
Blood agar
-5% sheep red blood cell for enrichment
-useful for growing Streptococci and fastidious organisms
-used to grow bacteria and identify pathogens
- used to test how bacteria break down red blood cells, which is called hemolysis.
-Testing bacteria for antibiotic susceptibility
Blood agar what the results look like
-Beta-hemolysis: A clear zone around the bacterial colonies
-Alpha-hemolysis: A green, opaque zone around the bacterial colonies
-Gamma-hemolysis: No notable zones around the bacterial colonies
Selective media
-growth medium that encourages the growth of specific microorganisms while inhibiting others
-composed of a basic medium to which antibiotics, chemicals, dyes, antiseptics, sodium salts, acid pH and other inhibitory substances that inhibit the growth of undesirable bacteria
-helps microbiologist isolate a specific bacterium in a mixed culture
-ex. Sabouraud Desxtrose agar, Colistin Nalidixic Acid agar, Thayer Martin agar (enriched and selective for Neisseria gonorrhoeae)
Differential media
-makes it easy to distinguish colonies of different microbes (based on biochemical properties)
-uses changes in color or appearance of the colonies to distinguish the bacteria from one another based on chemical reactions such as fermentation
-ex. Blood agar
-distinguish bacterial species that lyse (breakdown- hemolysis) sheep red blood cells
-sheep red cells are an enrichment- often added to culture media like blood agar plates to provide essential nutrients and factors that support the growth of fastidious (delicate) bacteria
-chocolate agar
How is chocolate agar made?
-made by heating defibrinated blood after it’s added to a blood agar base.
-The heating process lyses the blood cells, releasing nutrients that bacteria need to grow.
-The medium also contains beef extract, which provides nitrogenous nutrients, vitamins, and mineral
Beta hemolysis
-Process that completely destroys RBCs
-Bacteria produce an enzyme called streptolysin, which causes beta-hemolysis.
-clear or transparent zone around bacterial colonies on a blood agar plate
Alpha hemolysis
-partial breakdown of red blood cells that produces a green or brown discoloration around bacterial colonies.
Gamma hemolysis
-describes bacteria that don’t cause any lysis or clearing of red blood cells (RBCs) when cultivated on blood agar
-“non-hemolytic”
the bacterium is gram positive Mesophilic, and favulatively halophic and can grow without oxygen. the cell are cocci in clusters
Staphylococcus
MacConkey Agar
-selective and differential agar
-selectively allows only Gram-negative bacteria to grow while differentiating them based on their ability to ferment lactose
-selective: presence of bile salts and crystal violet in MacConkey agar inhibits the growth of Gram-positive bacteria, making it selective for Gram-negative organisms.
-selective agents: bile salts and crystal violet
-differential: inclusion of lactose and a pH indicator (neutral red) allows for differentiation between Gram-negative bacteria based on their ability to ferment lactose (further differentiate between gram-(-) bacteria based on their lactose metabolism). Lactose fermenting bacteria produce acidic byproducts which turn the colonies pink on MacConkey agar, while non-lactose fermenters appear colorless.
-differential agent: lactose
-color change indicator: neutral red
Mannitol salt agar
-selective and differential agar
-selective: 7.5% salt concentration inhibits most organisms and supports Staphylococcus
-favors bacteria that can survive high salt concentrations
-differential: sugar mannitol, which bacteria can use as an energy source. Bacteria that ferment mannitol produce acidic byproducts that lower the pH of the agar.
-pH indicator: phenol red- turns yellow when the pH decreases due to acid production from mannitol fermentation.
-bacteria that ferment mannitol will produce a yellow halo around their colonies, while those that cannot will not change the color of the media.
As a nurse in a plastic surgery clinic, you instruct pt’s on post-op care of their sutures. You tell pt’s before rem’ing bandages, to wash gently around the surgical site w/ soap and water and to swab the would w/ hydrogen peroxide. One day a pt called alarmed that the hydrogen peroxide caused her wound to bubble. What would you tell the pt?
Humans have the enzyme catalase which breaks down hydrogen peroxide into water and molecular oxygen. The oxygen is causing the bubbles. The bubbles loosen debris and promotes healing.
Capnophiles
-require ⬆️CO2
-optimal CO2 level: 5%
-⬇️O2
-ex. Pathogenic Neisseria, Harmophilus
Capnophiles procedure
-candle jar
-container that uses a lit candle to create an environment w/ ⬆️CO2 and ⬇️O2
1)Place a lit candle in a jar with a tight-fitting lid
2)Seal the lid
3)The candle burns until it goes out due to lack of oxygen
4)The jar is now filled with a carbon dioxide-rich, oxygen-poor atmosphere
-CO2 packet
-CO2 incubator
Anaerobic culture methods
used to cultivate anaerobic organisms (microbes that grow without oxygen) by creating an oxygen-free environment.
1) anaerobic jar- This environment is achieved by using a gas pack system or an indicator strip that contains chemicals which react to form hydrogen gas and a vacuum to remove residual oxygen
2) candle jar- used to create a microaerophilic environment by placing a lit candle inside a sealed jar, which consumes the oxygen within, leaving a higher concentration of carbon dioxide
3) cell culture- methods used to grow cells outside of their natural environment in a laboratory. These techniques involve isolating and expanding specific cells, and then maintaining them in culture vessels.
Cell culture
-the process of growing cells from an organism in a controlled laboratory environment in or on a cultured medium
-Cell cultures can be used to study cell behavior, develop drugs, and diagnose infections
-viruses” and “intracellular bacteria” refer to microorganisms that can replicate inside host cells, meaning they need to be grown within a living cell line in order to study their behavior and propagation
How does cell culture work?
-Cells are removed from an organism, either directly from tissue or from a cell line
-The cells are grown in a controlled environment with a suitable medium, temperature, and pH
-medium provides nutrients like amino acids, vitamins, and minerals
-Gases like oxygen and carbon dioxide, as well as other factors like osmotic pressure, also play a role in cell growth
Biosafety levels
1)no special precautions
2)lab coat, gloves, eye protection
3)Biosafety cabinets to prevent airborne transmission
4)sealed, (-)pressure “hot zone”
-exhaust air is filtered twice through HEPA filters
Pure culture
-contains only one species or strain
-a colony is a pop. of cells arising from a single cell or spore or from a group of attached cells
-“colony-forming unit” (CFU)
Binary fission
1) cell elongates and DNA is replicated
2) cell wall and plasma membrane begin to constrict
3) cross-wall forms, completely separating the 2 DNA copies
4) cells separate
cell division can be considered a logarithmic progression because each cell division results in a doubling of the cell population, which is a characteristic of logarithmic growth
Generation time
-time required for the cell to divide
-most bacteria have a generation time of 1-3hrs
-in E. coli doubling occurs every 20 mins
Bacterial growth curve
1) lag phase: intense activity preparing for pop. growth, but no ⬆️ in pop.
2) log phase: logarithmic or exponential ⬆️ in pop. (Due to reproduction via binary fission (bacteria) or mitosis (yeast)
3) stationary phase: period of equilibrium; microbial deaths balance production of new cells
4) death phase: ⬇️pop. at logarithmic rate
Cells are most metabolically active during which phase?
Log phase
Explain how and why the pH of culture media is controlled?
Most bacteria grow between pH 6.5 and 7.5. When bacteria are cultured in the laboratory, they often produce acids that eventually interfere with their own growth. To Neutralize the acids and maintain the proper pH, chemical buffers are included in the growth medium. The peptones and amino acids in some media act as buffers, and many media also contain phosphate salts. Phosphate salts have the advantage of exhibiting their buffering effect in the pH growth range of most bacteria
Explain the importance of osmotic pressure to microbial growth
- Growth of cell is inhibited if high concentration occurs. High salt and sugar concentrations draw water out any microbial cells that are present and thus prevent their growth
Compare and contrast growth of anaerobes and microaerophiles
anaerobes cannot tolerate any oxygen and are harmed by its presence, whereas microaerophiles require a low level of oxygen for growth and are inhibited by high oxygen concentrations
Describe the formation of biofilms and their potential for causing infection
biofilm is a complex community of microorganisms that adhere to a surface and are encased within a self-produced sticky matrix called extracellular polymeric substances (EPS), which can lead to infections by creating a protective barrier that makes the bacteria within highly resistant to antibiotics and immune system attacks; this often occurs on medical devices like catheters, implants, and dental surfaces, leading to chronic infections that are difficult to treat.
Proper specimen collection
collecting the correct type and amount of specimen in the right container, and then labeling and transporting it to a lab.
Colony
visible mass of microorganisms all originating from a single mother cell
Inoculum
substance that is introduced into a medium to start a culture or infection. It can be a population of cells, viruses, bacteria, or spores.
Streak plate technique
1) Sterilize the inoculating loop.
2) Gently touch the loop to the sample to collect cells.
3) Using a zigzag motion, spread the cells across the agar plate.
4) Repeat the process, moving to a new quadrant of the plate and using a fresh, sterile loop each time.
Lag phase
-Bacteria adapt to their environment and prepare for reproduction
-There is little to no cell reproduction
-The number of ribosomes per cell and the translation rate increase
Exponential (log) phase
-Bacteria divide rapidly by binary fission
-The number of cells increases exponentially
-Bacteria are most susceptible to antibiotics and disinfectants
-Cells have a constant growth rate and uniform metabolic activity
Stationary phase
rate of cell division equals the rate of cell death, Resources become limited, and The population stabilizes.
Death (decline) phase
-The bacterial population declines
-The rate of cell death is greater than the rate of cell growth
-Factors like nutrient depletion or environmental stress contribute to the decline
Direct methods of measuring cell growth
Direct Methods: Plate counts, Filtration, MPN, and Direct microscopic count.
1) Plate counts: A method of determining the number of bacteria in a sample by counting the number of colony-forming units on a solid culture medium. It measures the number of viable cells.
2) Filtration: The passage of a liquid or gas through a screenlike material, a filter removes most bacteria.
3) Most probable number method (MPN): A statistical determination of the number of coliforms per 100 ml of water or 100g of food.
4) Direct microscopic count: Enumeration of cells by observation through a microscope.
Indirect methods of measuring cell growth
Indirect Methods: Turbidity, Metabolic activity, and Dry weight
Turbidity: cloudiness. As bacteria multiply in a liquid medium, it becomes turbid, or cloudy with cells. A spectrophometer is used.
Metabolic activity: This method assumes that the amount of a certain metabolic product in in direct proportion to the number of bacteria present. Ex: acid production is used to determine amounts of vitamins.
Dry weight: In this procedure, the fungus is removed from the growth medium, filtered to remove extraneous material, and dried in a desiccator. It is then weighed. For bacteria, the same basic procedure is followed.
Collision nalidixic acid agar
selects for gram-(+) bacteria in a mixed culture
Sabouraud dextrose agar
Selects for fungi
Chemically defined media
One in which the exact chemical composition is known
Complex media
One in which the exact chemical composition varies slightly from batch to batch
Catalase
Catalyzes the breakdown of H2O2 and H2O
