Microbial Growthpdf

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Chemical Requirements Carbon. Besides water, which is needed because nutrients must be in solution in order to be used, car-

bon is a primary requirement for growth. It is the structural backbone of living matter. Its valence of fourallows it to be used in constructing complicated organic molecules. Sources of carbon are carbon dioxideor organic materials.

Nitrogen, Sulfur, and Phosphorus. Some organisms use proteinaceous material as a nitrogensource; others use ammonium ions (NH 4 + ) or nitrate ions (NO 3 ). A few bacteria and Cyanobacteria areable to use gaseous nitrogen (N 2 ) directly from the atmosphere. This process is called nitrogen xation.The Rhizobium and Bradyrhizobium bacteria, in symbiosis with leguminous plants, also x nitrogen. Sulfursources are sulfate ion (SO 4 2 ), hydrogen sulde (H 2 S), and the sulfur-containing amino acids. An impor-tant source of phosphorus is the phosphate ion (PO 4 3 ). Nitrogen and sulfur are required to synthesizeproteins. DNA, RNA, and ATP require nitrogen and phosphorus.

Trace Elements. Mineral elements such as iron, copper, molybdenum, and zinc are referred to as traceelements. Although sometimes added to laboratory media, they usually are assumed to be naturallypresent in water and other media components.

Oxygen. Microbes that use molecular oxygen are aerobes; if oxygen is an absolute requirement,they are obligate aerobes. Facultative anaerobes use oxygen when it is present but continue growth by fermentation or anaerobic respiration when it is not available. Facultative anaerobes grow moreefciently aerobically than they do anaerobically. Obligate anaerobes are bacteria totally unable touse oxygen for growth and usually nd it toxic. Hydrogen atoms in the electron transport chainmay be passed to oxygen, forming toxic hydrogen peroxide (H 2 O2 ). Aerobic organisms usually pro-duce catalase, an enzyme that breaks down hydrogen peroxide to water and oxygen; anaerobes usu-ally lack catalase. Aerotolerant anaerobes cannot use oxygen for growth but tolerate it fairly well.They will grow on the surface of a solid medium without the special techniques required forcultivation of less oxygen-tolerant anaerobes. Common examples of aerotolerant anaerobes are the

bacteria that ferment carbohydrates to lactic acid, a process that occurs in making many fermentedfoods. A few bacteria are microaerophilic, meaning they grow only in oxygen concentrations lowerthan that found in air. They are aerobic, however, in the sense that they require oxygen. They are

probably unusually sensitive to superoxide free radicals and peroxides, which they produce underoxygen-rich conditions.Oxygen has a number of toxic forms.

1. Singlet oxygen is normal molecular oxygen (O 2 ) that has been boosted into a higher energy stateand is extremely reactive.

2. Superoxide free radicals (O2 ) are formed in small amounts by aerobic organisms; they are so toxicthat the bacteria must neutralize them with superoxide dismutase. This enzyme converts superox-ide free radicals into oxygen and toxic hydrogen peroxide (which contains the peroxide anion ),which in turn is converted into oxygen and water by the enzyme catalase: 2 H 2 O2 2 H 2 O + O 2 .Another enzyme that breaks down hydrogen peroxide is peroxidase. It does not produce oxygen:H 2 O2 + 2 H + 2 H 2 O. Anaerobic bacteria often cannot neutralize the superoxide free radicals theyproduce and do not tolerate atmospheric oxygen.

3. The hydroxyl radical (OH ) is formed in cytoplasm by ionizing radiation and as a by-product of aer-obic respiration. It is probably the most reactive form.

Organic Growth Factors. Organic growth factors are organic compounds such as vitamins, aminoacids, and pyrimidines that are needed for life, but that a given organism is unable to synthesize.

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Culture MediaAny nutrient material prepared for the growth of bacteria in a laboratory is called a culture medium.Microbes growing in a container of culture medium are referred to as a culture. When microbes areadded to initiate growth, they are an inoculum. To ensure that the culture will contain only the microor-ganisms originally added to the medium (and their offspring), the medium must initially be sterile.

When a solid medium is required, a solidifying agent such as agar is added. Agar is a polysaccharidederived from a marine seaweed. Few microbes can degrade agar, so it remains a solid. It melts at aboutthe boiling point of water but remains liquid until the temperature drops to about 40C.

In a chemically dened medium, the exact chemical composition is known.Most heterotrophic bacteria and fungi are routinely grown on complex media, in which the exact

chemical composition varies slightly from batch to batch. Complex media are made up of nutrients suchas extracts from yeasts, beef, or plants, or digest of proteins from these and other sources. In many of these media, the energy, carbon, nitrogen, and sulfur requirements of the microorganisms are largelymet by partially digested protein products called peptones. Vitamins and other organic growth factorsare provided by meat extracts or yeast extracts. Such extracts supplement the organic nitrogen and car-

bon compounds but mainly are sources of soluble vitamins and minerals. This type of medium in liquidform is nutrient broth; when agar is added, it is nutrient agar.

Anaerobic Growth Media and Methods Obligately anaerobic bacteria often require reducing media for isolation. Because oxygen may be lethal,these media contain ingredients, such as sodium thioglycolate, that chemically combine with dissolvedoxygen to deplete the oxygen content of the culture medium. Obligate anaerobes may be grown on thesurface of solid media in anaerobic atmospheres produced in special jars in which an oxygen-freeatmosphere is generated by a chemical reaction. It is also possible to handle anaerobic organisms inanaerobic glove boxes lled with inert gases and tted with airtight rubber glove arms and air locks. Anew technique in which an enzyme, oxyrase, is added to the growth medium transforms the Petri plate(OxyPlate) into a self-contained anaerobic chamber.

Special Culture Techniques A few bacteria have never been grown successfully on articial laboratory media; the leprosy andsyphilis organisms are examples. Obligate intracellular parasites such as rickettsias and chlamydias alsodo not ordinarily grow on articial media. They, like viruses, require a living host cell. Carbon dioxideincubators, candle jars, and plastic bags with self-contained chemical gas generators are used to grow

bacteria with special CO 2 concentration requirements. Capnophiles are microbes that grow better athigh CO 2 concentrations.

Selective and Differential Media Selective media are designed to suppress the growth of unwanted bacteria and encourage the growthof the desired microorganisms. Antibiotics, high concentrations of salt, or high acidity might be used.Differential media make it easier to distinguish colonies of the desired organism from other coloniesgrowing on the same plate. The colonies have different colors or cause different changes in thesurrounding medium. Sometimes selective and differential functions are combined in the one medium.

Enrichment Culture Because bacteria may be present only in small numbers and may be missed, and because the bacteriumto be isolated may be of an unusual physiological type, it is sometimes necessary to resort to enrich-ment culture, or enrichment media. The conditions or sole nutrient are designed to increase small num-

bers of a certain organism to a detectable level.

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Obtaining Pure Cultures There are several methods for isolating bacteria in pure cultures, which contain only one kind of organism.

Streak Plate Method. Probably the most common method of obtaining pure cultures is the streakplate (Figure 6.1). A sterile inoculating needle is dipped into a mixed culture and streaked in a patternover the surface of the nutrient medium. The last cells rubbed from the needle are wide enough apartthat they grow into isolated visible masses called colonies.

Preserving Bacterial CulturesIn deep-freezing, a pure culture of microbes is placed in a suspending liquid and quick-frozen at50 to 95C. In lyophilization (freeze-drying), a suspension of microbes is quickly frozen at tempera-tures of 54 to 72C and the water removed by a high vacuum. The remaining powder can be storedfor many years and the surviving microorganisms cultured by hydrating them with a suitable liquidnutrient medium.

Growth of Bacteria CulturesBacterial DivisionBacteria normally reproduce by binary ssion. Genetic material becomes evenly distributed; then atransverse wall is formed across the center of the cell, and it separates into two cells. A few bacterialspecies reproduce by budding; that is, an initial outgrowth enlarges to cell size and then separates.Some lamentous species produce reproductive spores or simply fragment into viable pieces.

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FIGURE 6.1 Streak plate method for isolation of pure cultures of bacteria. (a) The direction of streaking is indicat-

ed by arrows. Streak series 1 is made from the original bacterial mixture. The inoculating loop is sterilized followingeach streak series. In series 2 and 3, the loop picks up bacteria from the previous series, diluting the number of cellseach time. There are numerous variants of such patterns. (b) In series 3 of this example, note that well-isolatedcolonies of two different types of bacteria have been obtained.

Colonies

(a) (b)

1

2

3


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Generation Time The time required for a cell to divide or the population to double is called generation (doubling) time.Bacterial populations are usually graphed logarithmically rather than arithmetically to permit the han-dling of the immense differences in numbers (Figure 6.2).

Phases of GrowthWhen bacterial population changes are graphed as a bacterial growth curve, certain phases becomeapparent. The lag phase shows little or no cell division. However, metabolic activity is intense. In thelog phase, the cells are reproducing most actively, and their generation time reaches a minimum andremains constant; a logarithmic plot produces an ascending straight line. They are then most activemetabolically and most sensitive to adverse conditions. In a chemostat, it is possible to keep a populationin such exponential growth indenitely. Without a chemostat, however, microbial deaths eventually

balance numbers of new cells, and a stationary phase is reached. When the number of deaths exceedsnumbers of new cells formed, the death phase, or logarithmic decline, is reached.

Measurement of Microbial Growth

Plate Counts. Dilutions of a bacterial suspension are distributed into a suitable solid nutrient medium by serial dilution, and the colonies appearing on the plates are counted (Figure 6.3). Colonies do notalways arise from single cells but from chains or clumps, so counts are often reported as colony-formingunits. The FDA convention is to count plates with 25250 colonies, but others may specify plates with30300 colonies.

Filtration. Bacteria may be sieved out of a liquid suspension onto a thin membrane lter with porestoo small for bacteria to pass. This lter can be transferred to a pad soaked in nutrient medium wherecolonies arise on the surface of the lter.

Microbial Growth 67

FIGURE 6.2 Growth curve for an exponentially increasing population, plotted logarithmically (dashed line)and arithmetically (solid line).

10,000

100,000

500,000

1,000,000(1,048,576)

(524,288)

(262,144)

(131,072)(65,536)

(32,768)(1024)(32)

(Log 10 = 1.51)

(Log 10 = 3.01)

(Log 10 = 4.52)

(Log 10 = 6.02)6.0

5.0

4.0

3.0

2.0

1.0

0

L o g 1 0 o

f n u m

b e r s o

f c e l l s

N u m

b e r s o

f c e

l l s

10 15 205Generations

0


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68 Chapter 6

(a)

(b)

FIGURE 6.3 Plate counts and serial dilutions. (a) In serial dilutions, the original inoculum is diluted in a seriesof dilution tubes. In our example, each succeeding dilution tube will have only one-tenth the number of microbial cells as the preceding tube. Then samples of the dilution are used to inoculate Petri plates, on which coloniesgrow and can be counted. This count is then used to estimate the number of bacteria in the original sample.(b) Methods for preparation of plates for plate counts.

(a) The pour plate method (b) The spread plate method

1 Inoculateempty plate

2 Add meltednutrient agar

3 Swirl to mix

4 Coloniesgrow in andon solidifiedmedium

1 Inoculate platecontainingsolid medium

2 Spread inoculumover surfaceevenly

3 Colonies growonly on surfaceof medium

Bacterial dilution

0.1 ml1.0 or 0.1 ml

Calculation: Number of colonies on plate reciprocal of dilution of sample = number of bacteria/ml

(For example, if 32 colonies were on a plate of1

/ 10,000 dilution, then the count is 32

10,000 = 320,000/ml in sample.)

1:10 1:100 1:1000 1:10,000 1:100,000Dilutions

9 ml brothin each tube

Originalinoculum

1 ml 1 ml 1 ml1 ml1 ml

1 ml

1:10 1:100 1:1000 1:10,000 1:100,000

1 ml 1 ml 1 ml 1 ml

Plating


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Most Probable Number (MPN). In the most probable number method, a sample is diluted out in aseries of tubes of liquid medium. The greater the number of bacteria, the more dilutions it takes to dilutethem out entirely and leave a tube without growth. Results of such dilutions can be compared to statisti-cal tables, and a cell count can be estimated.

Direct Microscopic Count. In this method, a measured volume of a bacterial suspension is placed

into a dened area on a special microscope slide, of which there are several designs. A microscope isused to count the cells in microscope elds. The average number per eld can be multiplied by a factorthat will estimate the total numbers.

Turbidity. To estimate turbidity, a beam of light is transmitted through a bacterial suspension to aphotoelectric cell. The more bacteria, the less light passes. This is recorded as absorbance (sometimescalled optical density, or OD) on the spectrophotometer or colorimeter.

Metabolic Activity. Microbial numbers can be estimated by the time required to produce acid orother products.

Dry Weight. Measuring weight is often the most satisfactory method for lamentous organisms suchas actinomycete bacteria or fungi.

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Self-TestsIn the matching section, there is only one answer to each question; however, the letteredoptions (a, b, c, etc.) may be used more than once or not at all.

I. Matchinga. Buffer

b. Mesophile

c. Thermophile

d. Psychrophile

e. Psychrotroph

f. Plasmolysis

g. Extreme halophile

h. Facultative halophile

i. Hyperthermophile

j. Capnophile

k. Facultative psychrophile

1. Adapted to high salt concentrations, which are required forgrowth.

2. The general term used for organisms capable of growthat 0C.

3. Capable of growth at high temperatures; optimum 50to 60C.

4. Used in media to neutralize acids.

5. A phenomenon that occurs when bacteria are placed inhigh salt concentration.

6. Term used in text for organisms that grow well at refriger-ator temperatures; optimum growth is at temperatures of 2030C.

7. Microbes that grow better at high CO 2 concentrations.

8. Members of the archaea with an optimum growth temper-

ature of 80C or higher.

9. Considered a synonym for psychrotroph by somemicrobiologists.

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II. Matching

III. Matchinga. Hydroxyl radical

b. Peroxidase

c. Superoxide dismutase

d. Superoxide free radicals

e. Singlet oxygen

f. Catalase

g. Oxyrase

1. Breaks down hydrogen peroxide without generation of oxygen.

2. Formed in cytoplasm by ionizing radiation.

3. An enzyme that converts hydrogen peroxide into oxygenand water.

4. The toxic form of oxygen neutralized by superoxidedismutase.

5. A component added to some culture media that makes thePetri plate into a self-contained anaerobic chamber.

a. Nitrogen xation

b. Obligate aerobe

c. Obligate anaerobe

d. Aerotolerant anaerobe

e. Catalase

f. Microaerophile

g. Peptones

h. Agar

1. An enzyme acting upon hydrogen peroxide.

2. Rhizobium bacteria do this in symbiosis with leguminousplants.

3. Requires atmospheric oxygen to grow.

4. Requires atmospheric oxygen, but in lower than normalconcentrations.

5. Does not use oxygen, but grows readily in itspresence.

6. Does not use oxygen and usually nds it toxic.

7. Important source of energy, carbon, nitrogen, and sulfur

requirements in complex media.

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IV. Matching

V. Matching

VI. Matchinga. Selective media

b. Differential media

c. Complex media

d. Reducing media

e. Chemically dened media

1. Used to grow obligate anaerobes.

2. Designed to suppress the growth of unwanted bacteria andto encourage growth of desired microbes.

3. Generally contain ingredients such as sodium thioglycolate

that chemically combine with dissolved oxygen.4. Nutrients are digests or extracts; exact chemical composi-

tion varies slightly from batch to batch.

a. Log phase

b. Lag phase

c. Death phase

d. Stationary phase

1. New cell numbers balanced by death of cells.

2. No cell division, but intense metabolic activity.

3. A logarithmic plot of the population produces an

ascending straight line.

a. Pour plate

b. Streak plate

c. Spread plate

d. Differential medium

e. Reducing medium

f. Enrichment culture

g. Lyophilization

h. Deep-freezing

i. Chemostat

j. Inoculum

k. Colonies

1. Isolation method for getting pure cultures; uses an inoculat-ing loop to trace a pattern of inoculum on a solid medium.

2. A device for maintaining bacteria in a logarithmic growth

phase.

3. Used to increase the numbers of a small minority of microorganisms in a mixed culture to arrive at a detectablelevel of microorganisms.

4. Preservation method that uses quick-freezing and a highvacuum.

5. Accumulations of microbes large enough to see without amicroscope.

6. Microbes added to initiate growth.

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18. The general term for tests that estimate microbial growth by the time required for them to deplete

oxygen in the medium is tests.

19. The growth temperature is that at which the organism grows best.

20. When a single colony arises from a clump of bacteria, it is recorded as a .

Label the Art

Critical Thinking1. What conditions that are characteristics of the food tend to retard spoilage in each of the following

foods?

a. Grape jelly

b. Pickles

c. Salted sh

d. Cheddar cheese

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I. II.

1

2

3

4

Cell wall Plasma membrane

DNA(nucleararea)

0 5 10Time (hr.)

L o g o

f n u m

b e r s o

f b a c

t e r i a

a.

b. c.d.


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2. What kinds of microorganisms (molds, lactic acid bacteria, endospore-forming bacteria, aerobic bacteria, etc.) would be most likely to cause spoilage of each of the foods listed on page 74? ( Hint:See Chapter 28.)

3. Plate counts are the most common method used to enumerate microbial populations. Discuss theadvantages or disadvantages of the use of plate counts for:

a. Milk intended for commercial sale

b. Molds

4. Draw a bacterial growth curve indicating the four phases of growth. At which phase of growthwould exposure to antibiotics cause the most adverse effects on the bacterial population? Why?

5. Complete the following table and indicate where the microorganisms will grow in a tube of a solidmedium on the basis of their relationship to oxygen.

Microbial Growth 75

Where in the tubeRelation to oxygen does growth occur? Why?

Obligate aerobe

Facultative anaerobe

Obligate anaerobe

Aerotolerant anaerobe

Microaerophile


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AnswersMatching

I. 1. g 2. d 3. c 4. a 5. f 6. e 7. j 8. i 9. kII. 1. e 2. a 3. b 4. f 5. d 6. c 7. g

III. 1. b 2. a. 3. f 4. d 5. g

IV. 1. b 2. i 3. f 4. g 5. k 6. jV. 1. d 2. b 3. aVI. 1. d 2. a 3. d 4. c

Fill in the Blanks1. polysaccharide 2. Cyanobacteria 3. mesophiles 4. number 5. broth 6. deep-freezing7. binary 8. absorbance (also optical density) 9. dry weight measurement 10. facultative11. facultative 12. phosphate; amino acids 13. culture medium 14. 40C 15. pour plate method16. peptones 17. living host cells 18. reduction 19. optimum 20. colony-forming units

Label the Art I. 1. Cell elongates and DNA divides 2. Cell wall and plasma membrane begin to divide 3. Crosswall forms completely around divided DNA 4. Cells separateII. a. Lag phase b. Log, or exponential growth, phase c. Stationary phase d. Death, or logarithmicdecline, phase

Critical Thinking1. a. Fruit jelly is acidic and also has a relatively high osmotic pressure from added sugars.

b. Pickles are acidic.c. Salted sh have high osmotic pressures.d. Hard cheeses are acidic and have relatively low moisture.

2. a. Molds; they are relatively tolerant of acidity, high osmotic pressure, and low moisture. (Yeastshave similar characteristics but are much less common in the environment. Acidophilic bacteriawill grow in acidic foods, but are usually not considered spoilage organismsin fact, they verylikely were used to make the food acidic, for example, cheese and pickles.)

b. Moldsc. Moldsd. Molds

3. a. Milk is highly perishable and the delay required for incubation of plates would often be toolengthy for practical use.

b. Molds are lamentous and plate counts would often arise from mold spores or fragments of mold laments, which would not indicate the mold growth very well.

4. See gure on page 77.Lag phaseThe period immediately following inoculation to fresh media in which little or nogrowth occurs. A time of intense metabolic activity as the cells gear up for reproduction.Log or exponential phaseThe period of growth in which cellular reproduction is most active.Generation time is at a minimum.Stationary phaseThe number of new cells being produced equals the number of cell deaths; theperiod of equilibrium.Death phaseThe number of dead cells exceeds the number of living cells until only a small por-tion of the population exists or the population dies out completely.

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Exposure to antibiotics during log or exponential phase would cause the most adverse effects on the bacterial population. This is because antibiotics are most effective against growing cells.

5.

0 5 10Time (hr)

L o g o

f n u m

b e r s o

f b a c

t e r i a

Lagphase

Log, orexponentialgrowth,phase

Stationary

phase Death, orlogarithmicdecline, phase

Microbial Growth 77

Where in the tubeRelation to oxygen does growth occur? Why?

Obligate aerobe Near the top of the Dissolved oxygen diffusesmedium only a short distance in

the medium

Facultative anaerobe Best near surface but They can survive withoutthroughout tube oxygen but grow better in

the presence of oxygen

Obligate anaerobe At the bottom Oxygen is poisonous tothese organisms

Aerotolerant anaerobe Evenly throughout tube These organisms dontuse oxygen

Microaerophile In a narrow band of the They will grow at themedium depth having the

optimum oxygenconcentration


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