The “minimum” and “maximum” are temperatures beyond which no growth takes place. The “optimum”
is the temperature at which growth rate is highest.
0
-10 10 20 30 40 50 60 70 80 90 100 110 120 Psychrophiles
Psychrotrophs Mesophiles
Thermophiles
Extreme Thermophiles
Temperature (oC) LogGrowthRate
2-40 THERMALCLASSIFICATIONS OFBACTERIA✦Refer to the text for a description of each categor y.
hot water bath, or
⽧cold water bath Per Student Group
✦twenty sterile Nutrient Broths
✦two Trypticase Soy Agar (TSA) plates
✦four sterile transfer pipettes
✦fresh Nutrient Broth cultures of:
⽧Escherichia coli
⽧Serratia marcescens
⽧Bacillus stearothermophilus
⽧Pseudomonas fluorescens
Procedure
Lab One
1Obtain 20 Nutrient Broths—one broth for each or- ganism at each temperature. Label them accordingly.
Also obtain two TSA plates and label them 20°C and 35°C, respectively.
2Mix each culture thoroughly before making the fol- lowing transfers. Using a sterile pipette, transfer a single dropof each broth culture to its appropriate Nutrient Broth tube. (Note:Because you will be comparing the amount of growth in the Nutrient Broth tubes, you must be sure to begin by transfer- ring the same volume of culture to each one. Use the same pipette for all transfers with a single organism.)
3Using a simple zigzag pattern (as in Exercise 1-4), inoculate each plate with Serratia marcescens.
4Incubate all tubes in their appropriate temperatures for 24 to 48 hours. Incubate the plates in the 20°C and 35°C incubators in an inverted position.
Lab Two
1Clean the outside of all tubes with a tissue, and place them in a test tube rack organized into groups by organism.
2Shake each broth gently until uniform turbidity is achieved.
3Compare all tubes in a group to each other. Rate each as 0, 1, 2, or 3, according to its turbidity (0 is clear and 3 is highly turbid). Record these in the Broth Data chart on the Data Sheet.
4Examine the plates incubated at different tempera- tures, compare the growth characteristics, and enter your results in the Plate Data chart on the Data Sheet.
5Using the data from the Broth data chart, determine the cardinal temperatures and classification of each of the four organisms.
6On the graph paper provided on the Data Sheet, plot the data (numeric values versus temperature) for the four organisms.
References
Forbes, Betty A., Daniel F. Sahm, and Alice S. Weissfeld. 2002. Chapters 2 and 10 in Bailey & Scott’s Diagnostic Micro biology, 11th ed. Mosby- Yearbook, St. Louis.
Holt, John G., Ed. 1994. Bergey’s Manual of Determinative Bacteriology, 9th ed. Williams and Wilkins, Baltimore.
Moat, Albert G., John W. Foster, and Michael P. Spector. 2002. Pages 597–601 in Microbial Physiology,4th ed. Wiley-Liss, New York.
Prescott, Lansing M., John P. Harley, and Donald A. Klein. 2005.
Chapter 6 in Microbiology, 6th ed., WCB McGraw-Hill, Boston.
Varnam, Alan H., and Malcolm G. Evans. 2000. Environmental Microbiology.ASM Press, Washington, DC.
White, David. 2000. Pages 384–387 in The Physiology and Biochemistry of Prokaryotes,2nd ed. Oxford University Press, New York.
Winn, Washington C., et al.2006. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology,6th ed. Lippincott Williams & Wilkins, Baltimore.
✦ Theory
The conventional means of expressing the concentration (or activity) of hydrogen ions in a solution is “pH”. The term pH, which stands for “pondus hydrogenii” (variably defined as hydrogen power or hydrogen potential), was invented in 1909 by the Danish biochemist, S쏗ren Peter Lauritz S쏗rensen. The 0–14 pH range he developed is a logarithmic scale designed to simplify acid and base cal- culations that otherwise would be expressed as molar values. S쏗rensen’s formula for the calculation of pH is expressed as follows:
pH ⳱– log [HⳭ]
For example, an aqueous solution containing 10–6moles of disassociated hydrogen ions per liter would be con- verted using S쏗rensen’s formula as follows:
pH ⳱– log [HⳭ] pH ⳱– log 10–6M HⳭ pH ⳱6
Pure water contains 10ⳮ7moles of hydrogen ions per liter and has a pH of 7. As hydrogen ions increase, the solution becomes more acidic and the pH decreases (see Table 2-1).
Bacteria live in habitats throughout the pH spectrum;
however, the range of most individual species is small.
Like temperature and salinity, pH tolerance is used as a means of classification. The three major classifications are
1. acidophiles: organisms adapted to grow well in environments below about pH 5.5,
2. neutrophiles: organisms that prefer pH levels between 5.5 and 8.5, and
3. alkaliphiles: organisms that live above pH 8.5.
Under normal circumstances, bacteria maintain a near-neutral internal environment regardless of their habitat; pH changes outside an organism’s range may destroy necessary membrane potential (in the produc- tion of ATP) and damage vital enzymes beyond repair.
This denaturingof cellular enzymes may be as minor as
2-10 The Effect of pH
on Microbial Growth
Solution
classification H+
Acidity/ Concentration Common Organismal
Alkalinity pH In Moles/Liter Examples classification
0 100 Nitric acid
1 10ⳮ1 Stomach acid
2 10ⳮ2 Lemon juice
3 10ⳮ3 Vinegar, cola
4 10ⳮ4 Tomatoes, orange juice
5 10ⳮ5 Black cof fee Acidophiles
Acidic 6 10ⳮ6 Urine
Neutral 7 10ⳮ7 Pure water Neutrophiles
Alkaline 8 10ⳮ8 Seawater
9 10ⳮ9 Baking soda Alkaliphiles
10 10ⳮ10 Soap, milk of magnesia
11 10ⳮ11 Ammonia
12 10ⳮ12 Lime water [Ca(OH)2]
13 10ⳮ13 Household bleach
14 10ⳮ14 Drain cleaner
TABLE2-1 PH SCALE
conformational changes in the proteins’ tertiary structure, but usually is lethal to the cell.
Acids from carbohydrate fermentation and alkaline products from protein metabolism are sufficient to dis- rupt microbial enzyme integrity when grown in vitro.
This is why buffers made from weak acids such as hydro- gen phosphate are added to bacteriological growth media.
In solution, buffers are able to alternate between weak acid (H2PO4
–) and conjugate base (HPO42–) to maintain HⳭ/OH–equilibrium.
HⳭⳭHPO42ⳮ➞H2PO4ⳮ
OHⳮⳭH2PO4ⳮ➞HPO42ⳮⳭH2O
✦ Application
This is a qualitative procedure used to estimate the minimum, maximum, and optimum pH for growth of a bacterial species.
✦ This Exercise
Today you will cultivate and observe the effects of pH on four organisms. Then you will classify them based on your results.
✦ Materials
Per Student Group
✦five of each pH adjusted Nutrient Broth as follows:
pH 2, pH 4, pH 6, pH 8, and pH 10
✦four sterile transfer pipettes
✦fresh Nutrient Broth cultures of:
⽧Lactobacillus plantarum
⽧Lactococcus lactis
⽧Enterococcus faecalis(BSL-2)
⽧Alcaligenes faecalis (BSL-2)
✦ Medium Recipe
pH-Adjusted Nutrient Broth
⽧Beef extract 3.0 g
⽧Peptone 5.0 g
⽧Distilled or deionized water 1.0 L
⽧NaOH or HCl as needed to adjust pH
Procedure
Lab One
1Obtain five tubes of each pH broth—one of each pH per organism (20 tubes total). Label them accordingly.
2Mix each culture thoroughly before making the following transfers. Using a sterile pipette, transfer a single dropof each broth culture to its appropriate Nutrient Broth tube. (Note:Because you will be comparing the amount of growth in the Nutrient Broth tubes, you must be sure to begin by trans - ferring the same volume of culture to each tube.
Use the same pipette for all transfers with a single organism.)
3Incubate all tubes at 35Ⳳ2°C for 48 hours.
Lab Two
1Clean the outside of all tubes with a tissue and place them in a test tube rack organized into groups by organism.
2Shake each broth gently until uniform turbidity is achieved.
3Compare all tubes in a group to each other. Rate each one as 0, 1, 2, or 3 according to its turbidity (0 is clear and 3 is highly turbid). Enter your obser- vations in the chart on the Data Sheet. (Note:Some color variability may exist between the different pH broths; therefore, base your conclusions solely on turbidity, not color.)
4Determine the range and classification of each test organism. Record the information on the Data Sheet.
5On the graph paper provided on the Data Sheet, plot the data (numeric values versus pH) of the four organisms.
References
Forbes, Betty A., Daniel F. Sahm, and Alice S. Weissfeld. 2002. Chapter 10 in Bailey & Scott’s Diagnostic Microbiology, 11th ed. Mosby, St. Louis.
Holt, John G., Ed. 1994. Bergey’s Manual of Determinative Bacteriology, 9th ed. Williams and Wilkins, Baltimore.
Varnam, Alan H., and Malcolm G. Evans. 2000. Environmental Micro - biology.ASM Press, Washington, DC.
Winn, Washington C., et al.2006. Koneman’s, Color Atlas and Textbook of Diagnostic Microbiology,6th ed. Lippincott Williams & Wilkins, Baltimore.
2-11 The Effect of Osmotic Pressure on Microbial Growth
Cell wall
Cell membrane
Hypotonic Isotonic Hypertonic
2-41 THEEFFECT OFOSMOTICPRESSURE ONBACTERIALCELLS✦This osmosis diagram illustrates the movement of water into and out of cells. The labels refer to the osmotic pressure of the solution outside the cell. In a hyposmotic environment, the cell has greater osmotic pressure, so the net movement of water (arrows) will be into the cell. In an isosmotic environment, there is no net movement because the osmotic pressure of the cell and that of the environment are equal. (Actually, the water is moving equally in both directions.) In a hyperosmotic environment, the osmotic pressure of the environment is greater, so the net movement is outward and results in plasmolysis. Note the shrinking membrane (CM) and the rigid cell wall (CW) in the hyperosmotic solution.
✦ Theory
Water is essential to all forms of life. It is not only the principal component of cellular cytoplasm, but also an essential source of electrons and hydrogen ions. Prokary- otes, like plants, require water to maintain cellular turgor pressure. Whereas eukaryotic cells burst with a constant influx of water, prokaryotes require water to prevent shrinking of the cell membrane, resulting in separation from the cell wall—an occurrence known as plasmolysis.
Many bacteria regulate turgor pressure by trans - porting in and maintaining a relatively high cytoplasmic potassium or sodium ion concentration, thereby creating a concentration gradient that promotes inward diffusion of water. For bacteria living in saline habitats, the job of maintaining turgor pressure is continuous because of the constant efflux of water.
Irrespective of a cell’s efforts to control its internal environment, natural forces will cause water to move through its semipermeable membrane from an area of low soluteconcentration to an area of high solute con- centration. In a solution in which solute concentration is low, water concentration is high, and vice versa. There- fore, water moves through a cell membrane from where its concentration is high to where its concentration is low. This process is called osmosis, and the force that controls it is calledosmotic pressure.
Osmotic pressure is a quantifiable term and refers, specifically, to the ability of a solution to pull water
toward itselfthrough a semipermeable membrane. If a bacterial cell is placed into a solution that is hyposmotic (a solution having low osmotic pressure),there will be a netmovement of water into the cell. If an organism is placed into a hyperosmoticsolution (a solution having high osmotic pressure), there will be a net movement of water out of the cell. For a bacterial cell in an isosmotic solution (a solution having osmotic pressure equal to that of the cell), water will tend to move in both directions equally; that is, there is no net movement (Figure 2-41).
Bacteria constitute a diverse group of organisms and, as such, have evolved many adaptations for survival.
Microorganisms tend to have a distinct range of salinities that are optimal for growth, with little or no survival outside that range. For example, some bacteria called halophilesgrow optimally in NaCl concentrations of 3% or higher. Extreme halophilesare organisms with specialized cell membranes and enzymes that require salt concentrations from 15% up to about 25% and will not survive where salinity is lower (Figure 2-42).
Except for a few osmotolerantbacteria, which will grow over a wide range of salinities, most bacteria live where NaCl concentrations are less than 3%.
✦ Application
This is a qualitative procedure used to demonstrate bacterial tolerances to NaCl.
✦ In This Exercise
You will be growing three microorganisms at a variety of NaCl concentrations to determine the tolerance range of salinities and optimum salinity for each organism.
✦ Materials
Per Student Group
✦four tubes each of saline medium prepared with 0%, 5%, 10%, 15%, 20%, and 25% NaCl
✦three sterile transfer pipettes
✦fresh cultures of:
⽧Escherichia coli
⽧Halobacterium
⽧Staphylococcus aureus(BSL-2)
✦ Medium Recipe
Modified Halobacterium Broth
⽧Sodium chloride 0 g, 50 g, 100 g, 150 g, 200 g, or 250 g
⽧Magnesium sulfate, heptahydrate 20.0 g
⽧Trisodium citrate, dihydrate 3.0 g
⽧Potassium chloride 2.0 g
⽧Casamino acids 5.0 g
⽧Yeast extract 5.0 g
⽧Deionized water 1.0 L
Adjust pH to 7.2 using 5 Mor concentrated HCl.
Procedure
Lab One
1 Obtain four tubes of each HalobacteriumBroth—
one of each concentration per organism plus a control. Label them accordingly.
2 Mix each culture thoroughly before making the following transfers. Using a sterile pipette, transfer a single dropof each broth culture to its appropriate HalobacteriumBroth tube. (Because you will be comparing the amount of growth in the Halobac- teriumBroth tubes, take care to begin by transferring the same volume of culture to each tube. Use the same pipette for all transfers with a single organism.)
3 Incubate all tubes at 35Ⳳ2°C for 48 hours.
Lab Two
1 Clean the outside of all tubes with a tissue, and place them in a test tube rack organized into groups by organism.
2 Shake each broth gently until uniform turbidity is achieved.
3 Compare all tubes in a group to each other and to the control. Rate each one as 0, 1, 2, or 3 according to its turbidity (0 is clear and 3 is very turbid.) (Note:
The different broths may show some color variability.
Check the uninoculated controls and, thus, base your conclusions solely on turbidity, not color.
4 Enter your results in the chart provided on the Data Sheet.
5 On the graph paper provided with the Data Sheet, plot the numeric value of turbidity versus the salt concentration for all three organisms.
References
Forbes, Betty A., Daniel F. Sahm, and Alice S. Weissfeld. 2002. Chapter 10 in Bailey & Scott’s Diagnostic Microbiology, 11th ed. Mosby, Inc., St.
Louis.
Hauser, Juliana T. 2006. Techniques for Studying Bacteria and Fungi.
Carolina Biological Supply Company, 2700 York Road, Burlington, NC.
Holt, John G., Ed. 1994. Bergey’s Manual of Determinative Bacteriology, 9th ed. Williams and Wilkins, Baltimore.
Koneman, Elmer W., Stephen D. Allen, William M. Janda, Paul C.
Schreckenberger, and Washington C. Winn, Jr. 1997. Color Atlas and Textbook of Diagnostic Microbiology, 5th ed. J. B. Lippincott Co., Philadelphia.
Moat, Albert G., John W. Foster, and Michael P. Spector. 2002. Pages 582–587 in Microbial Physiology, 4th ed. Wiley-Liss, New York.
Varnam, Alan H., and Malcolm G. Evans. 2000. Environmental Micro - biology.ASM Press, Washington, DC.
White, David. 2000. Pages 388–394 in The Physiology and Biochemistry of Prokaryotes, 2nd ed. Oxford University Press, New York.
2-42 A SALTERN INSANDIEGOBAY✦Salterns are low pools of saltwater used in the har vesting of salt. As water evaporates, the saltwater becomes saltier and saltier, until only salt remains.
This can then be purified and sold. The colors in the pools result from dif ferent communities of halophilic microorganisms that are associated with different salinities as the pools dr y out.
Control of Pathogens: Physical and Chemical Methods
Ever y patient in a hospital or other clinical setting has the right to expect that he or she will not contract a disease or infection while in that institution’s care. Ever y person donating blood at a blood bank or mobile center has the right to expect that all materials and sur faces they come in contact with will be free of pathogens. Workers in health clinics, hospitals, medical laboratories, and public health laboratories have the right to assume that reasonable precautions have been and are being taken to protect their safety while in the workplace.
These are only a few of the many reasons why the impor tance of understanding and use of pathogen control systems cannot be overemphasized. For tunately, with relatively few exceptions, the above- described conditions exist in this and other developed countries largely because of the dedication of thousands of employees and the oversight of dozens of international, governmental, and private organiza- tions such as the World Health Organization (WHO), Centers For Disease Control and Prevention (CDC), Food and Drug Administration (FDA), Environmental Protection Agency (EPA), American Public Health Association (APHA), and Association of Official Analytical Chemists (AOAC). These and many other federal and private organizations are responsible for the proper testing, registration, and classification of the substances or systems used to prevent the spread of pathogens.
These substances or systems, both chemical and physical, are referred to broadly as germicides.
Some germicides are specific in nature and typically include the name of the target pathogen, such as
“tuberculocide,” “virucide,” or “sporocide.” Most germicides are broad-spectrum and, thus, target a wide variety of pathogens. Although some overlap occurs, germicidal systems fall into three categories:
decontamination, disinfection, or sterilization.
1. Decontaminationis the lowest level of control and is defined as “reduction of pathogenic micro - organisms to a level at which items are safe to handle without protective attire.” Decontamination usually includes physical cleaning with soaps or detergents, and removal of all (ideally) or most organic and inorganic material. Proper cleaning of all instruments and sur faces is considered the critical first step toward dis infection or sterilization because, to be fully effective, a disinfectant or sterilant must come in direct contact with all pathogens present. Materials left to dr y on a sur face or apparatus can actually shield pathogens from a disinfecting or sterilizing agent or other wise neutralize it.
2. Disinfection is the next level of control and is divided into three sublevels—low, medium, and high—
based on effectiveness against specific control pathogens or their surrogates. All sublevels kill large numbers, if not all, of the targeted pathogens but typically do not kill large numbers of spores. Some high-level disinfectants are called chemical sterilants because they have the ability to kill all vegetative cells and spores.
Disinfectants typically are liquid chemical agents but can also be solid or gaseous. Other dis infec- tion methods include dr y heat, moist heat, and ultraviolet light. Disinfectants that are designed to reduce or eliminate pathogens on or in living tissue are called antiseptics. For obvious safety reasons, antiseptics are subject to additional testing to minimize the risks of side-effects. Some antiseptics are considered drugs and, therefore, are regulated by the FDA.
3. Sterilizationis the complete elimination of viable organisms including spores and, as such, is the highest level of pathogen control. Sterilization can be achieved by some chemicals, some gases, incineration, dr y heat, moist heat, ethylene oxide gas, ionizing radiation (Gamma, X-ray, and electron- beam), low-temperature plasma (utilizing a combination of chemical sterilants and ultraviolet radiation in a vacuum chamber), or low-temperature ozone (utilizing bottled oxygen, water, and electricity in a chamber to produce a lethal level of ozone).
In this unit you will examine both physical and chemical means of pathogen control. The following exercises illustrate the germicidal effects of UV radiation, disinfection, antisepsis, and steam (moist heat) sterilization. (For more information on microbial control, refer to Exercise 1-1 GloGerm, and to Exercise 7-3 Antimicrobial Susceptibility Test.)✦
✦ Theory
Of the many methods or agents that have been developed for sterilizing surgical and dental instruments, microbio- logical media, infectious waste, and other materials not harmed by moisture or heat, steam is still the most effec- tive and most common. The device used most commonly for this purpose is called a steam sterilizer, or autoclave.
Autoclaves are relatively safe, easy to operate, and, if used properly, effective at killing all microbial vegetative cells and spores.
Under atmospheric pressure, water boils at 100ºC (212ºF). At pressures above atmospheric pressure, water must be heated above 100ºC before it will boil. Much like home pressure cookers, which create pressure and high temperatures to shorten cooking times, autoclaves use super-heated steam under pressure to kill heat- resistant organisms. Examples of heat-resistant organisms include members of the spore-producing genera—Bacillus, Geobacillus, andClostridium.
In the microbiology laboratory, sterilizing tempera- ture usually is set at between 121° and 127°C (250° and 260°F); however, sterilizing time can vary according to the size and consistency of the material being sterilized.2 At a minimum, to be sure that all vegetative cells and spores have been killed, items being processed must reach optimum temperature for at least 15 minutes. This includes items deep inside the autoclave container that may be partially insulated from the steam by surrounding items. Understandably, larger loads take longer to process than smaller loads do. (Certain sensitive appli cations, such as microbiological media preparation, in which formula integrity must be maintained and when specific growth inhibiting ingredients are included, lower times and temperatures are acceptable.)
To maintain laboratory safety and comply with laws regarding infectious waste disposal, sterilizers must be checked regularly for operating effectiveness. Special thermometers placed in an autoclave can record the
maximum temperature reached inside the chamber but do not measure how low the temperature dips during the normal cycling of the heating elements. Specialized color-coded autoclave tape can be a fairly good indi cator that sterilization is complete, but the only way, with certainty, to determine that sterilization has been achieved is by using a device called a biological indicator(Figure 2-43).
Biological indicators, as the name suggests, are test systems that contain something living. A typical biological indicator that is particularly useful for testing autoclaves is one that contains bacterial spores. Bacterial spores, the dormant form of an organism, are highly resistant to
2-12 Steam Sterilization 1
1Steam sterilization is not yet considered reliable at inactivating prion pro- teins, such as those that cause Bovine Spongiform Encephalopathy, the so-called Mad Cow Disease, and its variant, Creutzfeldt-Jakob disease.
2In clinics, hospitals, or other locations where surgical instruments are being processed, the World Health Organization (WHO) recommends a minimum processing time and temperature of 134°C for 18 minutes.
2-43 AUTOCLAVEBIOLOGICALINDICATORS✦These indicator vials contain an ampule of fermentation broth and a filter paper strip containing spores of Geobacillus stearothermophilus—a spore-forming organism capable of withstanding high tempera- tures. The vial in the center was autoclaved for 15 minutes at 121°C, cooled, pinched to crush the inner glass ampule, and incubated. The purple color (compared to the uncrushed nega- tive control on the right) indicates that an acidic condition from fermentation does not exist. This suggests that the organism has been killed by the autoclaving. Note the gray-colored band on the label. This chemical indictor changes from blue to gray upon autoclaving. The ampule in the negative control was not crushed, so the spores in the filter paper never made contact with the broth and, thus, provides a color example of unfer- mented broth. The ampule on the left is a positive control to verify the viability of the organism used in the system. It was not autoclaved (as evidenced by the blue band on the label) but was crushed and incubated. The de velopment of yellow color indicates that the organism in the system is viable and that the lack of yellow color in the center vial is a result of autoclaving.