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Microbiological Tests
A P P E N D I X
II Microbiological Tests David I. Prangnell*, Tzachi M. Samocha† *Texas Parks and Wildlife Department, San Marcos, TX, United States † Marine Solutions and Feed Technology, Spring, TX, United States
II.A VIBRIO MONITORING Vibrio spp. are gram-negative, facultative anaerobic, chemoautotrophic bacteria. They can grow under aerobic or anaerobic conditions in the presence of inorganic ions serving as electron acceptors, such as oxygen, nitrate, and sulfate. Regular monitoring of Vibrio spp. allows time to prevent a disease outbreak and test the effectiveness of probiotic treatments. Vibrio concentration is monitored with thiosulphate-citrate-bile salt sucrose (TCBS) agar (see Appendix IIb for a detailed method). TCBS has a high pH (8.5–9.5) that suppresses growth of most non-Vibrio bacteria, so it is highly selective for Vibrio. Sucrose-fermenting Vibrio produce yellow colonies and nonsucrose-fermenting Vibrio produce green colonies (Table AII.1 and Fig. AII.1—Appendix VII). Yellow colonies generally are nonpathogenic; green colonies are considered pathogenic to shrimp. Vibriosis often is observed in intensive closed systems when green-colony Vibrio species, particularly V. parahaemolyticus, increase relative to yellow-colony species (Fig. AII.1). Some pathogenic species, however, such as V. harveyi, V. alginolyticus, and V. campbelli, also may express
yellow colonies (Doug Ernst, Natural Shrimp, personal communication). V. harveyi and V. splendidus also can exhibit variations in color on TCBS, that is, yellow or green (Jeffrey Turner, TAMU-CC, personal communication). Some probiotic species also can form as yellow colonies on TCBS, and Pseudomonas spp. and Aeromonas spp. occasionally form blue-green colonies. The method described in Appendix IIb can be used for CHROMagar Vibrio plates (also known as RambaCHROM Vibrio), with colonies appearing as mauve (V. parahaemolyticus), green-blue to turquoise-blue (V. vulnificus and V. cholerae), or white (colorless) (V. alginolyticus) (Fig. AII.2). CHROMagar can be more specific for Vibrio than TCBS (Di Pinto et al., 2011), although it covers fewer species. A detailed biochemical key for Vibrio identification is found in Noguerola and Blanch (2008). Ray et al. (2010) describe three other methods for monitoring microbial communities in biofloc systems: visual microscopy abundance quantification, epifluorescence microscopy with image analysis quantification, and bacterial fatty acid assessment by gas chromatography. Black colonies growing on TCBS indicate sulfate-reducing bacteria. This indicates that
359
TABLE AII.1 Colony Color Formed by Different Pathogenic Vibrio spp. on TCBS Agar Plates According to Sucrose (Yellow) or Nonsucrose Fermenting (Green) (Noguerola and Blanch, 2008; Doug Ernst, personal communication; Jeffrey Turner, TAMU-CC, personal communication) Vibrio sp.
Colony Color
% Colonies Forming Color
V. alginolyticus
Yellow
75–89
V. anguillarum
Yellow
90
V. campbelli
Green
90
V. cholerae
Yellow
90
V. damsela
Green
–
V. fluvialis
Yellow
90
V. furnissii
Yellow
90
V. harveyi
Green (often with a lighter halo); luminescence
90
V. hepatarius
Yellow
90
V. metschnikovii
Yellow
90
V. mimicus
Green
90
V. mytili
Yellow
90
V. nereis
Yellow
90
V. nigrapulchritudo
Green
90
V. pacinii
Yellow
90
V. parahaemolyticus
Bluish-green
90
V. penaeicida
Green
90 (Poor growth on TCBS)
V. ponticus
Yellow
75–89
V. splendidus (I)
Yellow
75–89 (Weak)
V. splendidus (II)
Green
90
V. tubiashi
Yellow
90
V. vulnificus
Green (can be yellowish)
75–89/90 depending on strain
FIG. AII.1 TCBS agar plates with Vibrio colonies. (A) Yellow (light gray in print version) dominant [only one green (dark gray in print version)], (B) Higher proportion of green colonies.
APPENDIX II MICROBIOLOGICAL TESTS
361
FIG. AII.2 A CHROMagar Vibrio agar (CHROMagar-France) with mauve (V. parahaemolyticus), green-blue (light gray in print version) to turquoise-blue (dark gray in print version) (V. vulnificus/V. cholerae), and white (colorless) (V. alginolyticus) colonies. (Alberto Lerner, CHROMagar, http://www.chromagar.com/. Used with permission.)
highly toxic hydrogen sulfide is being generated, usually from an area of accumulated sludge. If this occurs, immediately raise DO, reduce feed ration, and—if it does not create another problem by raising un-ionized ammonia to unsafe levels—increase pH (Panakorn, 2016; Bob Rosenberry, personal communication). Maintain adequate mixing to avoid areas of sludge accumulation where anoxic conditions promote H2S production.
II.B TCBS PLATE TESTING METHOD FOR VIBRIO (Based on communication)
Doug
Ernst,
personal
Equipment • Sample bottles • Spray bottle containing a liquid surface disinfectant (e.g., 90% ethanol) • Hand disinfectant • Deionized water • Blender (hand-held or bench-top) • Micropipette and tips • Inoculating loop
• Bunsen burner or alcohol lamp • TCBS agar plates (Thiosulfate citrate bilesalts sucrose agar) (or more specific equivalent such as CHROMagar) • Incubation oven • Black fine tip marker Method Disinfect all work surfaces and equipment in the fume hood or sterile cabinet. Apply a liquid surface disinfectant, such as 90% ethanol, with a spray bottle. Wash hands thoroughly with freshwater and an alcohol-based hand disinfectant. 1. Collect water samples (200 mL) in labeled sterilized bottles. Sterilize the bottles with liquid disinfectant, rinse with deionized water and allow to air dry (preferably in an oven at >105°C) prior to collecting samples. Once samples are collected, rinse the outside of the sample bottles with freshwater and spray with surface disinfectant prior to placing on the workbench. 2. Label Petri dish covers with the sample ID and volume inoculated using a black fine tip marker.
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APPENDIX II MICROBIOLOGICAL TESTS
3. Blend the sample for 20 s to release the Vibrio cells from solids, either in the sample bottle or a disinfected container. Disinfect the blender and container with liquid disinfectant and rinse thoroughly with deionized water between samples. 4. Apply water samples to the plates using a micropipette with disposable tips. Target 30–300 colonies per plate for ease of counting. Apply a volume of 100 μL if the Vibrio count is expected to be <3000 cfu/mL. Apply 10 μL if the Vibrio count is expected to be >3000 cfu/mL. Inoculate replicate plates for a few samples to check results. 5. Place the sample drop on the plate while keeping the lid mostly covering the plate. Only open the lid for as short a time as needed to complete inoculation. Let the drop flow down one direction on one side of the plate, keeping it at least 1 cm away from the inside edges. Heat the inoculating loop in the flame (until red hot) and let it cool in air or in the sterile agar before using. Gently distribute the sample on the agar surface with the inoculating loop by using multiple parallel strokes in a perpendicular direction, working first in one direction and then perpendicular to the original direction, and finally in a third direction, covering the entire plate surface. Keep the sample away from the edges of the Petri dish. Just touch the surface of the agar without digging in. Resterilize the inoculating loop after each use. Bacterial plating methods and diagrams are available on the internet. 6. Incubate the plates for 18–24 h at 30–32°C (Matching culture tank temperature). 7. Perform plate counts immediately after removal from the incubator as some colonies
may change color at room temperature. Hold the plate upside down over or up to a light source. Count the colonies, using a black fine tip marker to mark the colonies on the underside of the plate as they are counted. 8. Report the number of yellow colonies, green colonies, and total colonies as cfu/mL and multiply the number by the dilution factor (10-μL sample 100, 100-μL sample 10). If the count is too high to reasonably count, divide the plate into equal segments (e.g., four quadrants), count the colonies on a portion of the segments (e.g., two quadrants), and multiply the result accordingly (e.g., by 2) for the total count. An example form and an Excel Sheet # 19 for recording Vibrio colony numbers are available in Page # 415 named Vibrio & Alkalinity Form_Examples & Calc Sheet—Appendix VII. The whole Vibrio monitoring process is shown in Video # 29—Appendix VIII.
References Di Pinto, A., Terio, V., Novello, L., Tantillo, G., 2011. Comparison between thiosulphate-citrate-bile salt sucrose (TCBS) agar and CHROMagar Vibrio for isolating Vibrio parahaemolyticus. Food Control 22 (1), 124–127. Noguerola, I., Blanch, A.R., 2008. Identification of Vibrio spp. with a set of dichotomous keys. J. Appl. Microbiol. 105, 175–185. Panakorn, S., 2016. Hydrogen sulfide—the silent killer. Aqua Culture Asia Pacific 12 (2), 14. Ray, A.J., Seaborn, G., Leffler, J.W., Wilde, S.B., Lawson, A., Browdy, C.L., 2010. Characterization of microbial communities in minimal-exchange, intensive aquaculture systems and the effects of suspended solids management. Aquaculture 310, 130–138.
II Microbiological Tests David I. Prangnell*, Tzachi M. Samocha† *Texas Parks and Wildlife Department, San Marcos, TX, United States † Marine Solutions and Feed Technology, Spring, TX, United States
II.A VIBRIO MONITORING Vibrio spp. are gram-negative, facultative anaerobic, chemoautotrophic bacteria. They can grow under aerobic or anaerobic conditions in the presence of inorganic ions serving as electron acceptors, such as oxygen, nitrate, and sulfate. Regular monitoring of Vibrio spp. allows time to prevent a disease outbreak and test the effectiveness of probiotic treatments. Vibrio concentration is monitored with thiosulphate-citrate-bile salt sucrose (TCBS) agar (see Appendix IIb for a detailed method). TCBS has a high pH (8.5–9.5) that suppresses growth of most non-Vibrio bacteria, so it is highly selective for Vibrio. Sucrose-fermenting Vibrio produce yellow colonies and nonsucrose-fermenting Vibrio produce green colonies (Table AII.1 and Fig. AII.1—Appendix VII). Yellow colonies generally are nonpathogenic; green colonies are considered pathogenic to shrimp. Vibriosis often is observed in intensive closed systems when green-colony Vibrio species, particularly V. parahaemolyticus, increase relative to yellow-colony species (Fig. AII.1). Some pathogenic species, however, such as V. harveyi, V. alginolyticus, and V. campbelli, also may express
yellow colonies (Doug Ernst, Natural Shrimp, personal communication). V. harveyi and V. splendidus also can exhibit variations in color on TCBS, that is, yellow or green (Jeffrey Turner, TAMU-CC, personal communication). Some probiotic species also can form as yellow colonies on TCBS, and Pseudomonas spp. and Aeromonas spp. occasionally form blue-green colonies. The method described in Appendix IIb can be used for CHROMagar Vibrio plates (also known as RambaCHROM Vibrio), with colonies appearing as mauve (V. parahaemolyticus), green-blue to turquoise-blue (V. vulnificus and V. cholerae), or white (colorless) (V. alginolyticus) (Fig. AII.2). CHROMagar can be more specific for Vibrio than TCBS (Di Pinto et al., 2011), although it covers fewer species. A detailed biochemical key for Vibrio identification is found in Noguerola and Blanch (2008). Ray et al. (2010) describe three other methods for monitoring microbial communities in biofloc systems: visual microscopy abundance quantification, epifluorescence microscopy with image analysis quantification, and bacterial fatty acid assessment by gas chromatography. Black colonies growing on TCBS indicate sulfate-reducing bacteria. This indicates that
359
TABLE AII.1 Colony Color Formed by Different Pathogenic Vibrio spp. on TCBS Agar Plates According to Sucrose (Yellow) or Nonsucrose Fermenting (Green) (Noguerola and Blanch, 2008; Doug Ernst, personal communication; Jeffrey Turner, TAMU-CC, personal communication) Vibrio sp.
Colony Color
% Colonies Forming Color
V. alginolyticus
Yellow
75–89
V. anguillarum
Yellow
90
V. campbelli
Green
90
V. cholerae
Yellow
90
V. damsela
Green
–
V. fluvialis
Yellow
90
V. furnissii
Yellow
90
V. harveyi
Green (often with a lighter halo); luminescence
90
V. hepatarius
Yellow
90
V. metschnikovii
Yellow
90
V. mimicus
Green
90
V. mytili
Yellow
90
V. nereis
Yellow
90
V. nigrapulchritudo
Green
90
V. pacinii
Yellow
90
V. parahaemolyticus
Bluish-green
90
V. penaeicida
Green
90 (Poor growth on TCBS)
V. ponticus
Yellow
75–89
V. splendidus (I)
Yellow
75–89 (Weak)
V. splendidus (II)
Green
90
V. tubiashi
Yellow
90
V. vulnificus
Green (can be yellowish)
75–89/90 depending on strain
FIG. AII.1 TCBS agar plates with Vibrio colonies. (A) Yellow (light gray in print version) dominant [only one green (dark gray in print version)], (B) Higher proportion of green colonies.
APPENDIX II MICROBIOLOGICAL TESTS
361
FIG. AII.2 A CHROMagar Vibrio agar (CHROMagar-France) with mauve (V. parahaemolyticus), green-blue (light gray in print version) to turquoise-blue (dark gray in print version) (V. vulnificus/V. cholerae), and white (colorless) (V. alginolyticus) colonies. (Alberto Lerner, CHROMagar, http://www.chromagar.com/. Used with permission.)
highly toxic hydrogen sulfide is being generated, usually from an area of accumulated sludge. If this occurs, immediately raise DO, reduce feed ration, and—if it does not create another problem by raising un-ionized ammonia to unsafe levels—increase pH (Panakorn, 2016; Bob Rosenberry, personal communication). Maintain adequate mixing to avoid areas of sludge accumulation where anoxic conditions promote H2S production.
II.B TCBS PLATE TESTING METHOD FOR VIBRIO (Based on communication)
Doug
Ernst,
personal
Equipment • Sample bottles • Spray bottle containing a liquid surface disinfectant (e.g., 90% ethanol) • Hand disinfectant • Deionized water • Blender (hand-held or bench-top) • Micropipette and tips • Inoculating loop
• Bunsen burner or alcohol lamp • TCBS agar plates (Thiosulfate citrate bilesalts sucrose agar) (or more specific equivalent such as CHROMagar) • Incubation oven • Black fine tip marker Method Disinfect all work surfaces and equipment in the fume hood or sterile cabinet. Apply a liquid surface disinfectant, such as 90% ethanol, with a spray bottle. Wash hands thoroughly with freshwater and an alcohol-based hand disinfectant. 1. Collect water samples (200 mL) in labeled sterilized bottles. Sterilize the bottles with liquid disinfectant, rinse with deionized water and allow to air dry (preferably in an oven at >105°C) prior to collecting samples. Once samples are collected, rinse the outside of the sample bottles with freshwater and spray with surface disinfectant prior to placing on the workbench. 2. Label Petri dish covers with the sample ID and volume inoculated using a black fine tip marker.
362
APPENDIX II MICROBIOLOGICAL TESTS
3. Blend the sample for 20 s to release the Vibrio cells from solids, either in the sample bottle or a disinfected container. Disinfect the blender and container with liquid disinfectant and rinse thoroughly with deionized water between samples. 4. Apply water samples to the plates using a micropipette with disposable tips. Target 30–300 colonies per plate for ease of counting. Apply a volume of 100 μL if the Vibrio count is expected to be <3000 cfu/mL. Apply 10 μL if the Vibrio count is expected to be >3000 cfu/mL. Inoculate replicate plates for a few samples to check results. 5. Place the sample drop on the plate while keeping the lid mostly covering the plate. Only open the lid for as short a time as needed to complete inoculation. Let the drop flow down one direction on one side of the plate, keeping it at least 1 cm away from the inside edges. Heat the inoculating loop in the flame (until red hot) and let it cool in air or in the sterile agar before using. Gently distribute the sample on the agar surface with the inoculating loop by using multiple parallel strokes in a perpendicular direction, working first in one direction and then perpendicular to the original direction, and finally in a third direction, covering the entire plate surface. Keep the sample away from the edges of the Petri dish. Just touch the surface of the agar without digging in. Resterilize the inoculating loop after each use. Bacterial plating methods and diagrams are available on the internet. 6. Incubate the plates for 18–24 h at 30–32°C (Matching culture tank temperature). 7. Perform plate counts immediately after removal from the incubator as some colonies
may change color at room temperature. Hold the plate upside down over or up to a light source. Count the colonies, using a black fine tip marker to mark the colonies on the underside of the plate as they are counted. 8. Report the number of yellow colonies, green colonies, and total colonies as cfu/mL and multiply the number by the dilution factor (10-μL sample 100, 100-μL sample 10). If the count is too high to reasonably count, divide the plate into equal segments (e.g., four quadrants), count the colonies on a portion of the segments (e.g., two quadrants), and multiply the result accordingly (e.g., by 2) for the total count. An example form and an Excel Sheet # 19 for recording Vibrio colony numbers are available in Page # 415 named Vibrio & Alkalinity Form_Examples & Calc Sheet—Appendix VII. The whole Vibrio monitoring process is shown in Video # 29—Appendix VIII.
References Di Pinto, A., Terio, V., Novello, L., Tantillo, G., 2011. Comparison between thiosulphate-citrate-bile salt sucrose (TCBS) agar and CHROMagar Vibrio for isolating Vibrio parahaemolyticus. Food Control 22 (1), 124–127. Noguerola, I., Blanch, A.R., 2008. Identification of Vibrio spp. with a set of dichotomous keys. J. Appl. Microbiol. 105, 175–185. Panakorn, S., 2016. Hydrogen sulfide—the silent killer. Aqua Culture Asia Pacific 12 (2), 14. Ray, A.J., Seaborn, G., Leffler, J.W., Wilde, S.B., Lawson, A., Browdy, C.L., 2010. Characterization of microbial communities in minimal-exchange, intensive aquaculture systems and the effects of suspended solids management. Aquaculture 310, 130–138.