Ch 6 & 7 - Microbial Growth and Control Stephanie Lanoue

Requirements for Growth Learning Objectives 6-1 Classify microbes into five groups on the basis of preferred temperature range. 6-2 Identify how and why the pH of culture media is controlled. 6-3 Explain the importance of osmotic pressure to microbial growth.

Environmental factors For Growth (Overview) Physical requirements __________________ _____ Osmotic pressure Chemical Requirements CHONPKS Trace elements-Fe, Cu, Zn, Mb Organic growth factors

Physical Requirements, Temperature Minimum growth temperature The _________ temperature at which the species will grow Optimum growth temperature Species grow _______ Maximum growth temperature The __________ temperature at which growth is possible

Physical Requirements, Temperature Temperature (optimal enzyme operation) Psychrophiles, _____-loving microbes (-10oC to 20o C) Mesophiles, __________ temperature-loving microbes (10o C to 50o C) Pathogens Thermophiles, _____-loving microbes, (40o C to 70o C) Extreme thermophiles (65o C to 110o C)

Physical Requirements, pH Most bacteria grow between pH ______ and 7.5 (best pH for most bacteria) Molds and yeasts grow between pH ____ and 6 Acidophiles grow in ______ environments pH 1.0 - 5.5 Ex. Lactobacillus acidophilus, H. pylori Alkalophiles pH 8.5 - 11.5 Ex. Bacillus alcalophilus

Physical Requirements, Osmotic Pressure (OP) Bacteria are better adapted to _____ OP High OP remove water from a cell Environment is hypertonic High ______ or sugar concentration in environment (solute) Plasmolysis occurs at high OP Shrinkage of cells Figure 6.4

Cell in isotonic solution. Under these Figure 6.4 Plasmolysis. Plasma membrane Plasma membrane Cell wall H2O Cytoplasm Cytoplasm NaCl 0.85% NaCl 10% Cell in isotonic solution. Under these Plasmolyzed cell in hypertonic solution. conditions, the solute concentration in the cell is equivalent to a solute concentration of 0.85% sodium chloride (NaCl). If the concentration of solutes such as NaCl is higher in the surrounding medium than in the cell (the environment is hypertonic), water tends to leave the cell. Growth of the cell is inhibited.

Requirements for Growth Learning Objectives 6-4 Name a use for each of the four elements (carbon, nitrogen, sulfur, and phosphorus) needed in large amounts for microbial growth. 6-5 Explain how microbes are classified on the basis of oxygen requirements. 6-6 Identify ways in which aerobes avoid damage by toxic forms of oxygen.

Chemical Requirements Carbon Structure of ________ molecules Most important requirements for microbial growth Nitrogen, sulfur, phosphorous To synthesize cellular material such as _____ and protein trace elements, small amount of other chemical such as, zinc Function of enzymes as cofactors We have talked about cofactors before in a previous chapter. What is a cofactor?

Chemical Requirements, Oxygen Obligate aerobes—_______ oxygen Facultative anaerobes—grow via fermentation or anaerobic respiration when oxygen is available Obligate anaerobes—grow occurs where there is ___ oxygen

Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria

Chemical Requirements, Organic Growth Factors Organic compounds obtained from the ____________ Vitamins, amino acids, purines, and pyrimidines

Culture Media Learning Objectives 6-8 Distinguish chemically defined and complex media. 6-9 Justify the use of each of the following: anaerobic techniques, living host cells, candle jars, selective and differential media, enrichment medium. 6-10 Differentiate biosafety levels 1, 2, 3, and 4.

Culture Media Agar __________ polysaccharide Used as a solidifying agent for culture media in Petri plates, slants, and deeps Generally ____ metabolized by microbes Liquefies at 100C Solidifies at ~40C https://learning.uonbi.ac.ke/courses/SBT202/scormPackages/path_2/culture_media.jpg

Culture Media https://learning.uonbi.ac.ke/courses/SBT202/scormPackages/path_2/culture_media.jpg

Culture Media Culture medium: nutrients prepared for microbial __________ Sterile: ___ living microbes Inoculum: ___________ of microbes into a medium Culture: microbes growing in or on a culture medium

Culture Media Chemically defined media: exact chemical composition is ________ Complex media: extracts and digests of yeasts, meat, or plants; chemical composition varies batch to batch Nutrient _______ Nutrient agar

Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia coli

Special Culture Techniques Biosafety levels BSL-1: __ special precautions; basic teaching labs BSL-2: lab coat, gloves, eye protection BSL-3: biosafety cabinets to prevent airborne transmission BSL-4: sealed, negative pressure; "hot zone" Exhaust air is filtered twice through HEPA filters

Media Types Selective, suppress growth of _________ bacteria Contain inhibitors to suppress growth Mannitol _______ agar : selective for halophiles with 7% salt (osmotic challenge) and differential for mannitol fermenters. Selects for Gram(+) and inhibits Gram(-) bacteria. Eosin Methylene Blue Agar (EMB) Agar: kills Gram(+) with eosin and methylene blue, selective for Gram(-). Differential for lactose fermenters. McConkey Agar: suppresses Gram(+) with crystal violet and bile salts and selects Gram(-). Differential for lactose fermenters.

Media Types (cont’d) Differential - distinguishes between ________ species (different microbes on the same plate) Blood agar (sheep’s blood) reveals if hemolytic

Media Types (cont’d) Enrichment - __________ nutrients to favor growth of special bacteria Lysed red blood cells provide unique nutrients in blood/chocolate agar

Obtaining Pure Cultures Learning Objectives 6-11 Define colony. 6-12 Describe how pure cultures can be isolated by using the streak plate method.

Pure Cultures • A pure culture contains only ____ species or strain • A colony is a ___________ of cells arising from a single cell or spore or from a group of attached cells • A colony is often called a colony-forming unit (CFU) The streak plate method is used to isolate pure cultures

The Growth of Bacterial Cultures Learning Objectives 6-14 Define bacterial growth, including binary fission. 6-15 Compare the phases of microbial growth, and describe their relation to generation time.

Bacterial Growth Refers to bacterial __________, not the cell size! Generation time, time required for cell ________ (1 hr to 3hr, for some 24hr) Growth Curve: Lag, Log, Stationary, Death Quantifying Growth If time 20 min, in 30 generation or 10 hr, population 1 billion

Bacterial Division, _______ fission

Generation Time Time required for a cell to ______ 20 minutes to 24 hours Binary fission _______ the number of cells each generation Total number of cells = 2number of generations Growth curves are represented logarithmically

Figure 6.13a Cell division.

Figure 6.13b Cell division.

Phases of Growth (In order) Log phase Stationary phase Death phase

Figure 6.15 Understanding the Bacterial Growth Curve.

The Growth of Bacterial Cultures Learning Objectives 6-16 Explain four direct methods of measuring cell growth. 6-17 Differentiate direct and indirect methods of measuring cell growth.

Measuring Bacterial Growth Direct measurement Plate _______ of viable bacterial forming colonies Direct microscopic count

Counts are performed on bacteria mixed into a dish with agar (pour plate method) or spread on the surface of a plate (spread plate method) After incubation, count colonies on plates that have 30-300 colonies (CFUs)

Plate counts Figure 6.17 Methods of preparing plates for plate counts. The pour plate method The pour plate method The spread plate method The spread plate method 1.0 or 0.1 ml 0.1 ml Inoculate empty plate. Inoculate plate containing solid medium. Bacterial dilution Spread inoculum over surface evenly. Add melted nutrient agar. Swirl to mix. Colonies grow only on surface of medium. Colonies grow on and in solidified medium. Plate counts

Direct Microscopic Count Volume of a bacterial suspension placed on a ________ Average _______ of bacteria per viewing field is calculated Uses a special Petroff-Hausser cell counter Number of bacteria/ml = Number of cells counted Volume of area counted

Grid with 25 large squares Figure 6.20 Direct microscopic count of bacteria with a Petroff-Hausser cell counter. Grid with 25 large squares Cover glass Slide Bacterial suspension is added here and fills the shallow volume over the squares by capillary action. Bacterial suspension Microscopic count: All cells in several large squares are counted, and the numbers are averaged. The large square shown here has 14 bacterial cells. Cover glass Slide Location of squares The volume of fluid over the large square is 1/1,250,000 of a milliliter. If it contains 14 cells, as shown here, then there are 14 × 1,250,000 = 17,500,000 cells in a milliliter. Cross section of a cell counter. The depth under the cover glass and the area of the squares are known, so the volume of the bacterial suspension over the squares can be calculated (depth × area).

Measuring by Indirect Methods Example: Turbidity—measurement of ___________ with a spectrophotometer More turbid = More bacteria Light source Spectrophotometer Light Blank Light-sensitive detector Scattered light that does not reach detector Bacterial suspension