[et_pb_section fb_built=\u00bb1″ _builder_version=\u00bb4.16″ global_colors_info=\u00bb{}\u00bb][et_pb_row module_class=\u00bb et_pb_row_fullwidth\u00bb _builder_version=\u00bb4.16″ width=\u00bb89%\u00bb width_tablet=\u00bb80%\u00bb width_last_edited=\u00bbon|desktop\u00bb max_width=\u00bb89%\u00bb max_width_tablet=\u00bb80%\u00bb max_width_last_edited=\u00bbon|desktop\u00bb custom_padding=\u00bb5px|0px|0|0px|false|false\u00bb make_fullwidth=\u00bbon\u00bb locked=\u00bboff\u00bb global_colors_info=\u00bb{}\u00bb column_structure=\u00bb2_3,1_3″ width_phone=\u00bb80%\u00bb max_width_phone=\u00bb80%\u00bb][et_pb_column type=\u00bb2_3″ _builder_version=\u00bb4.16″ custom_padding=\u00bb|||\u00bb global_colors_info=\u00bb{}\u00bb custom_padding__hover=\u00bb|||\u00bb][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb|700|||||||\u00bb text_text_color=\u00bb#256168″ text_font_size=\u00bb44px\u00bb text_line_height=\u00bb1.1em\u00bb text_font_size_tablet=\u00bb\u00bb text_font_size_phone=\u00bb33px\u00bb text_font_size_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n
Detection and enumeration methods<\/p>\n
[\/et_pb_text][et_pb_text _builder_version=\u00bb4.23.1″ text_font=\u00bb||||||||\u00bb text_text_color=\u00bb#256168″ text_font_size=\u00bb25px\u00bb text_line_height=\u00bb1.1em\u00bb background_color=\u00bb#eaeddb\u00bb custom_margin=\u00bb|||\u00bb custom_padding=\u00bb60px|60px|60px|95px|true\u00bb custom_padding_tablet=\u00bb60px|50px|60px|50px|true|true\u00bb custom_padding_phone=\u00bb40px|50px|40px|50px|true|true\u00bb custom_padding_last_edited=\u00bbon|tablet\u00bb hover_enabled=\u00bb0″ text_font_size_tablet=\u00bb\u00bb text_font_size_phone=\u00bb20px\u00bb text_font_size_last_edited=\u00bbon|phone\u00bb locked=\u00bboff\u00bb global_colors_info=\u00bb{}\u00bb sticky_enabled=\u00bb0″]<\/p>\n
4.1<\/strong> Methods based on direct observation of host cell lysis<\/a> [\/et_pb_text][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_line_height=\u00bb1.3em\u00bb header_font=\u00bb||||||||\u00bb header_2_font=\u00bb|700|||||||\u00bb header_2_text_color=\u00bb#0a2d31″ header_2_font_size=\u00bb33px\u00bb text_font_size_tablet=\u00bb\u00bb text_font_size_phone=\u00bb18px\u00bb text_font_size_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n Before starting to look at detection and enumeration, we should perhaps talk a little bit about sampling and conservation of the samples<\/strong>. When taking a sample the ideal situation would probably be to do the analyses as soon as possible, but the tests can be delayed<\/strong>, if needed, by 48-72 hours<\/strong> as long as the samples are kept at 4\u00baC<\/strong>. It is easy to concentrate phages from volumes up to one liter, and small volumes can keep their phage density for months<\/strong> when stored at -20\u00baC, or -80\u00baC after adding 10% v\/v glycerol.<\/strong><\/p>\n [\/et_pb_text][et_pb_button button_url=\u00bbhttp:\/\/www.ub.edu\/ubtv\/cerca\/?cercar=bacteriophage\u00bb url_new_window=\u00bbon\u00bb button_text=\u00bbaccess video content\u00bb button_alignment=\u00bbleft\u00bb _builder_version=\u00bb4.16″ custom_padding=\u00bb10px|50px|10px|50px|true|true\u00bb global_colors_info=\u00bb{}\u00bb button_text_size__hover_enabled=\u00bboff\u00bb button_one_text_size__hover_enabled=\u00bboff\u00bb button_two_text_size__hover_enabled=\u00bboff\u00bb button_text_color__hover_enabled=\u00bboff\u00bb button_one_text_color__hover_enabled=\u00bboff\u00bb button_two_text_color__hover_enabled=\u00bboff\u00bb button_border_width__hover_enabled=\u00bboff\u00bb button_one_border_width__hover_enabled=\u00bboff\u00bb button_two_border_width__hover_enabled=\u00bboff\u00bb button_border_color__hover_enabled=\u00bboff\u00bb button_one_border_color__hover_enabled=\u00bboff\u00bb button_two_border_color__hover_enabled=\u00bboff\u00bb button_border_radius__hover_enabled=\u00bboff\u00bb button_one_border_radius__hover_enabled=\u00bboff\u00bb button_two_border_radius__hover_enabled=\u00bboff\u00bb button_letter_spacing__hover_enabled=\u00bboff\u00bb button_one_letter_spacing__hover_enabled=\u00bboff\u00bb button_two_letter_spacing__hover_enabled=\u00bboff\u00bb button_bg_color__hover_enabled=\u00bboff\u00bb button_one_bg_color__hover_enabled=\u00bboff\u00bb button_two_bg_color__hover_enabled=\u00bboff\u00bb][\/et_pb_button][\/et_pb_column][et_pb_column type=\u00bb1_3″ _builder_version=\u00bb4.16″ custom_padding=\u00bb|||\u00bb global_colors_info=\u00bb{}\u00bb custom_padding__hover=\u00bb|||\u00bb][et_pb_image src=\u00bbhttps:\/\/coliphages.ixole.es\/wp-content\/uploads\/2018\/09\/cta-bluephage.jpg\u00bb url=\u00bbhttp:\/\/bluephage.com\/\u00bb url_new_window=\u00bbon\u00bb align_tablet=\u00bbcenter\u00bb align_last_edited=\u00bbon|desktop\u00bb _builder_version=\u00bb4.16″ global_colors_info=\u00bb{}\u00bb align_phone=\u00bbcenter\u00bb][\/et_pb_image][\/et_pb_column][\/et_pb_row][et_pb_row module_class=\u00bb et_pb_row_fullwidth\u00bb _builder_version=\u00bb4.16″ width=\u00bb89%\u00bb width_tablet=\u00bb80%\u00bb width_last_edited=\u00bbon|desktop\u00bb max_width=\u00bb89%\u00bb max_width_tablet=\u00bb80%\u00bb max_width_last_edited=\u00bbon|desktop\u00bb make_fullwidth=\u00bbon\u00bb global_colors_info=\u00bb{}\u00bb width_phone=\u00bb80%\u00bb max_width_phone=\u00bb80%\u00bb][et_pb_column type=\u00bb4_4″ _builder_version=\u00bb4.16″ custom_padding=\u00bb|||\u00bb global_colors_info=\u00bb{}\u00bb custom_padding__hover=\u00bb|||\u00bb][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_line_height=\u00bb1.2em\u00bb text_font_size_tablet=\u00bb\u00bb text_font_size_phone=\u00bb18px\u00bb text_font_size_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n When working with bacteriophages, we do not need to worry about phenomena such as stress, injury or reactivation that frequently lead to misinterpretation of environmental data on bacterial indicators.<\/p>\n All of the above makes it easy to work with phages as an indicator.<\/p>\n [\/et_pb_text][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_font_size=\u00bb28px\u00bb text_line_height=\u00bb1.3em\u00bb background_color=\u00bb#f0f1ec\u00bb custom_margin=\u00bb|||||true\u00bb custom_padding=\u00bb50px|50px|70px|50px||true\u00bb custom_padding_tablet=\u00bb\u00bb custom_padding_phone=\u00bb50px|30px|50px|30px|true|true\u00bb custom_padding_last_edited=\u00bbon|phone\u00bb text_font_size_tablet=\u00bb\u00bb text_font_size_phone=\u00bb21px\u00bb text_font_size_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n The detection and enumeration of an infectious<\/strong> (living) bacteriophage depends on the fact that they lyse a susceptible host cell<\/strong>.<\/p>\n [\/et_pb_text][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_line_height=\u00bb1.3em\u00bb header_font=\u00bb||||||||\u00bb header_2_font=\u00bb|700|||||||\u00bb header_2_text_color=\u00bb#0a2d31″ header_2_font_size=\u00bb33px\u00bb text_font_size_tablet=\u00bb\u00bb text_font_size_phone=\u00bb18px\u00bb text_font_size_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n \u00a0It is possible to detect the presence of phage genomes or fragments of genome by nucleic acid amplification techniques such as PRC. However, the detection of whole genomes<\/strong> or fragments of genome in a sample does not guarantee that they correspond to phages which are still alive<\/strong> and hence capable of infecting and replicating in a susceptible host cell.<\/p>\n Lysis<\/strong> of a host cell can be detected by<\/strong> visual observation due to the decrease in opacity<\/strong> of a matrix containing the host cells. This can be seen either by the appearance of clear areas<\/strong> in an opaque solid layer<\/strong> of host cells (see figures 1 and 2) or by a decrease in the optical density<\/strong> of a liquid suspension<\/strong> of host cells.<\/p>\n It is also possible to detect host cell lysis<\/strong> by detecting the appearance of an intracellular enzyme in the supernatant that has been released by lysis<\/strong>.<\/p>\n [\/et_pb_text][et_pb_text _builder_version=\u00bb4.23.1″ text_font=\u00bb||||||||\u00bb text_line_height=\u00bb1.3em\u00bb header_font=\u00bb||||||||\u00bb header_2_font=\u00bb|700|||||||\u00bb header_2_text_color=\u00bb#0a2d31″ header_2_font_size=\u00bb33px\u00bb hover_enabled=\u00bb0″ text_font_size_tablet=\u00bb\u00bb text_font_size_phone=\u00bb18px\u00bb text_font_size_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb module_id=\u00bb4-1″ sticky_enabled=\u00bb0″]<\/p>\n Nowadays, standardized methods for the detection and enumeration of bacteriophages are based on this principle. They can be quantitative<\/strong> methods and presence\/absence<\/strong> methods (Adams, 1959).<\/p>\n [\/et_pb_text][\/et_pb_column][\/et_pb_row][et_pb_row module_class=\u00bb et_pb_row_fullwidth\u00bb _builder_version=\u00bb4.16″ background_color=\u00bb#f0f1ec\u00bb width=\u00bb89%\u00bb width_tablet=\u00bb80%\u00bb width_last_edited=\u00bbon|desktop\u00bb max_width=\u00bb89%\u00bb max_width_tablet=\u00bb80%\u00bb max_width_last_edited=\u00bbon|desktop\u00bb custom_padding=\u00bb|||\u00bb make_fullwidth=\u00bbon\u00bb global_colors_info=\u00bb{}\u00bb column_structure=\u00bb1_2,1_2″ width_phone=\u00bb80%\u00bb max_width_phone=\u00bb80%\u00bb][et_pb_column type=\u00bb1_2″ _builder_version=\u00bb4.16″ custom_padding=\u00bb|||\u00bb global_colors_info=\u00bb{}\u00bb custom_padding__hover=\u00bb|||\u00bb][et_pb_image src=\u00bbhttps:\/\/coliphages.ixole.es\/wp-content\/uploads\/2018\/09\/01detection-ok.png\u00bb force_fullwidth=\u00bbon\u00bb align_tablet=\u00bbcenter\u00bb align_last_edited=\u00bbon|desktop\u00bb _builder_version=\u00bb4.16″ custom_margin=\u00bb30px|0px||0px||true\u00bb custom_margin_tablet=\u00bb\u00bb custom_margin_phone=\u00bb0px|||\u00bb custom_margin_last_edited=\u00bbon|phone\u00bb custom_padding=\u00bb|||30px\u00bb custom_padding_tablet=\u00bb\u00bb custom_padding_phone=\u00bb0px|20px|0px|20px|true|true\u00bb custom_padding_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb align_phone=\u00bbcenter\u00bb][\/et_pb_image][\/et_pb_column][et_pb_column type=\u00bb1_2″ _builder_version=\u00bb4.16″ custom_padding=\u00bb|||\u00bb global_colors_info=\u00bb{}\u00bb custom_padding__hover=\u00bb|||\u00bb][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_font_size=\u00bb18px\u00bb text_line_height=\u00bb1.3em\u00bb custom_margin=\u00bb20px|40px|20px||true\u00bb custom_margin_tablet=\u00bb\u00bb custom_margin_phone=\u00bb|20px||20px||true\u00bb custom_margin_last_edited=\u00bbon|phone\u00bb text_font_size_tablet=\u00bb\u00bb text_font_size_phone=\u00bb15px\u00bb text_font_size_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n Quantitative methods<\/strong> are those that allow direct quantification<\/strong> of infectious viral particles in a sample<\/strong>. The number of viruses is visible when making a culture consisting of an agar<\/strong> monolayer containing both bacteria<\/strong> and the sample<\/strong> that may contain bacteriophages. After incubation<\/strong> on a Petri plate, we can observe clear areas of different sizes where turbidity<\/strong> (a typical feature of Petri plates where there is a mass bacterial culture) is lost due to cell destruction<\/strong>. Each clear area (called a plaque)<\/strong> is caused by a bacteriophage<\/strong> (or clump of bacteriophages). When counting<\/strong>, we speak about plaque-forming units (PFUs)<\/strong> instead of saying an absolute number of viruses as it is possible that the virions attach to one another, and thus an agglomerate of more than one bacteriophage can only attack one bacterium. In some literature PFUs can also be called PFPs (plaque-forming particles).<\/p>\n [\/et_pb_text][et_pb_button button_url=\u00bbhttps:\/\/www.youtube.com\/watch?v=ddbaul2ijw8″ url_new_window=\u00bbon\u00bb button_text=\u00bbaccess video content\u00bb button_alignment=\u00bbcenter\u00bb _builder_version=\u00bb4.16″ custom_margin=\u00bb||10px||false\u00bb custom_padding=\u00bb10px|50px|10px|50px|true|true\u00bb custom_padding_tablet=\u00bb\u00bb custom_padding_phone=\u00bb|30px||30px||true\u00bb custom_padding_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb button_text_size__hover_enabled=\u00bboff\u00bb button_one_text_size__hover_enabled=\u00bboff\u00bb button_two_text_size__hover_enabled=\u00bboff\u00bb button_text_color__hover_enabled=\u00bboff\u00bb button_one_text_color__hover_enabled=\u00bboff\u00bb button_two_text_color__hover_enabled=\u00bboff\u00bb button_border_width__hover_enabled=\u00bboff\u00bb button_one_border_width__hover_enabled=\u00bboff\u00bb button_two_border_width__hover_enabled=\u00bboff\u00bb button_border_color__hover_enabled=\u00bboff\u00bb button_one_border_color__hover_enabled=\u00bboff\u00bb button_two_border_color__hover_enabled=\u00bboff\u00bb button_border_radius__hover_enabled=\u00bboff\u00bb button_one_border_radius__hover_enabled=\u00bboff\u00bb button_two_border_radius__hover_enabled=\u00bboff\u00bb button_letter_spacing__hover_enabled=\u00bboff\u00bb button_one_letter_spacing__hover_enabled=\u00bboff\u00bb button_two_letter_spacing__hover_enabled=\u00bboff\u00bb button_bg_color__hover_enabled=\u00bboff\u00bb button_one_bg_color__hover_enabled=\u00bboff\u00bb button_two_bg_color__hover_enabled=\u00bboff\u00bb][\/et_pb_button][\/et_pb_column][\/et_pb_row][et_pb_row module_class=\u00bb et_pb_row_fullwidth\u00bb _builder_version=\u00bb4.16″ width=\u00bb89%\u00bb width_tablet=\u00bb80%\u00bb width_last_edited=\u00bbon|desktop\u00bb max_width=\u00bb89%\u00bb max_width_tablet=\u00bb80%\u00bb max_width_last_edited=\u00bbon|desktop\u00bb make_fullwidth=\u00bbon\u00bb global_colors_info=\u00bb{}\u00bb width_phone=\u00bb80%\u00bb max_width_phone=\u00bb80%\u00bb][et_pb_column type=\u00bb4_4″ _builder_version=\u00bb4.16″ custom_padding=\u00bb|||\u00bb global_colors_info=\u00bb{}\u00bb custom_padding__hover=\u00bb|||\u00bb][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_line_height=\u00bb1.2em\u00bb text_font_size_tablet=\u00bb\u00bb text_font_size_phone=\u00bb18px\u00bb text_font_size_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n The two main quantitative methods<\/strong> are single agar layer (SAL)<\/strong> and double agar layer (DAL)<\/strong>. Both SAL and DAL are based on the appearance of plaques<\/strong> on an agar layer made of semisolid agar, a culture containing the desired bacterial strain, and the problem sample. After incubation overnight<\/strong> the plaques can be observed in the agar plate if there are bacteriophages in the sample. The difference between the two methods is that DAL has an extra solid agar layer under the semisolid<\/strong> one that makes a more stable base for the second layer, which is an advantage in situations where the Petri plates are in a bad state or made of poor quality materials. For the results obtained to be statistically valid<\/strong>, the number of PFUs on a Petri plate<\/strong> has to be between 30 and 300<\/strong>. This is why it is recommendable to make<\/strong> several dilutions<\/strong> of the sample before starting the procedure.<\/p>\n [\/et_pb_text][\/et_pb_column][\/et_pb_row][et_pb_row module_class=\u00bb et_pb_row_fullwidth\u00bb _builder_version=\u00bb4.16″ background_color=\u00bb#f0f1ec\u00bb width=\u00bb89%\u00bb width_tablet=\u00bb80%\u00bb width_last_edited=\u00bbon|desktop\u00bb max_width=\u00bb89%\u00bb max_width_tablet=\u00bb80%\u00bb max_width_last_edited=\u00bbon|desktop\u00bb custom_padding=\u00bb|||0px\u00bb make_fullwidth=\u00bbon\u00bb locked=\u00bboff\u00bb global_colors_info=\u00bb{}\u00bb column_structure=\u00bb1_2,1_2″ width_phone=\u00bb80%\u00bb max_width_phone=\u00bb80%\u00bb][et_pb_column type=\u00bb1_2″ _builder_version=\u00bb4.16″ custom_padding=\u00bb|||\u00bb global_colors_info=\u00bb{}\u00bb custom_padding__hover=\u00bb|||\u00bb][et_pb_image src=\u00bbhttps:\/\/coliphages.ixole.es\/wp-content\/uploads\/2018\/08\/02detection.png\u00bb force_fullwidth=\u00bbon\u00bb align_tablet=\u00bbcenter\u00bb align_last_edited=\u00bbon|desktop\u00bb _builder_version=\u00bb4.16″ custom_margin=\u00bb|||0px\u00bb custom_margin_tablet=\u00bb\u00bb custom_margin_phone=\u00bb\u00bb custom_margin_last_edited=\u00bbon|phone\u00bb custom_padding=\u00bb|20px||20px\u00bb global_colors_info=\u00bb{}\u00bb align_phone=\u00bbcenter\u00bb][\/et_pb_image][\/et_pb_column][et_pb_column type=\u00bb1_2″ _builder_version=\u00bb4.16″ custom_padding=\u00bb|||\u00bb global_colors_info=\u00bb{}\u00bb custom_padding__hover=\u00bb|||\u00bb][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_font_size=\u00bb18px\u00bb text_line_height=\u00bb1.3em\u00bb custom_margin=\u00bb20px|40px|20px||true\u00bb custom_margin_tablet=\u00bb\u00bb custom_margin_phone=\u00bb|20px||20px||true\u00bb custom_margin_last_edited=\u00bbon|phone\u00bb text_font_size_tablet=\u00bb\u00bb text_font_size_phone=\u00bb15px\u00bb text_font_size_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n The presence\/absence methods<\/strong> are those that, by themselves, only indicate<\/strong> whether or not there are bacteriophages<\/strong> in a given volume of the tested sample<\/strong>. The traditional presence\/absence method is the spot test<\/strong>, which consists of putting a drop<\/strong> of a decontaminated aliquot of an enrichment culture<\/strong> on a Petri plate where previously we cultured bacteria. After incubating<\/strong>, the place where the drop was put loses its turbidity due to cell destruction if there are bacteriophages<\/strong> in the sample. The enrichment culture consists of a liquid culture of the host bacteria, inoculated with an aliquot of a sample and incubated for a number of hours.<\/p>\n [\/et_pb_text][et_pb_button button_url=\u00bbhttps:\/\/www.youtube.com\/watch?v=W8nOklhhEw4″ url_new_window=\u00bbon\u00bb button_text=\u00bbAccess video content\u00bb button_alignment=\u00bbcenter\u00bb _builder_version=\u00bb4.16″ custom_padding=\u00bb10px|50px|10px|50px|true|true\u00bb custom_padding_tablet=\u00bb\u00bb custom_padding_phone=\u00bb|20px||20px||true\u00bb custom_padding_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb button_text_size__hover_enabled=\u00bboff\u00bb button_one_text_size__hover_enabled=\u00bboff\u00bb button_two_text_size__hover_enabled=\u00bboff\u00bb button_text_color__hover_enabled=\u00bboff\u00bb button_one_text_color__hover_enabled=\u00bboff\u00bb button_two_text_color__hover_enabled=\u00bboff\u00bb button_border_width__hover_enabled=\u00bboff\u00bb button_one_border_width__hover_enabled=\u00bboff\u00bb button_two_border_width__hover_enabled=\u00bboff\u00bb button_border_color__hover_enabled=\u00bboff\u00bb button_one_border_color__hover_enabled=\u00bboff\u00bb button_two_border_color__hover_enabled=\u00bboff\u00bb button_border_radius__hover_enabled=\u00bboff\u00bb button_one_border_radius__hover_enabled=\u00bboff\u00bb button_two_border_radius__hover_enabled=\u00bboff\u00bb button_letter_spacing__hover_enabled=\u00bboff\u00bb button_one_letter_spacing__hover_enabled=\u00bboff\u00bb button_two_letter_spacing__hover_enabled=\u00bboff\u00bb button_bg_color__hover_enabled=\u00bboff\u00bb button_one_bg_color__hover_enabled=\u00bboff\u00bb button_two_bg_color__hover_enabled=\u00bboff\u00bb][\/et_pb_button][\/et_pb_column][\/et_pb_row][et_pb_row module_class=\u00bb et_pb_row_fullwidth\u00bb _builder_version=\u00bb4.16″ width=\u00bb89%\u00bb width_tablet=\u00bb80%\u00bb width_last_edited=\u00bbon|desktop\u00bb max_width=\u00bb89%\u00bb max_width_tablet=\u00bb80%\u00bb max_width_last_edited=\u00bbon|desktop\u00bb make_fullwidth=\u00bbon\u00bb global_colors_info=\u00bb{}\u00bb width_phone=\u00bb80%\u00bb max_width_phone=\u00bb80%\u00bb][et_pb_column type=\u00bb4_4″ _builder_version=\u00bb4.16″ custom_padding=\u00bb|||\u00bb global_colors_info=\u00bb{}\u00bb custom_padding__hover=\u00bb|||\u00bb][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_line_height=\u00bb1.3em\u00bb header_font=\u00bb||||||||\u00bb header_2_font=\u00bb|700|||||||\u00bb header_2_text_color=\u00bb#0a2d31″ header_2_font_size=\u00bb33px\u00bb text_font_size_tablet=\u00bb\u00bb text_font_size_phone=\u00bb18px\u00bb text_font_size_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n This kind of method does not allow direct quantification, but the number of infectious viral particles can be determined using quantal (statistics-based) methods<\/strong>. The most used of them is MPN (most probable number)<\/strong> which consists of making presence\/absence tests with different amounts (or dilutions) of the sample and making several replicas of each amount (or dilution) of sample to determine, using statistics, the amount of virus in a certain volume of sample<\/strong>. This kind of method increases their sensitivity when increasing the number of replicas performed, until the point that they can be as accurate as quantitative methods.<\/p>\n [\/et_pb_text][et_pb_button button_url=\u00bbhttps:\/\/www.youtube.com\/user\/melodyscrudato\/videos?shelf_id=0&view=0&sort=dd\u00bb url_new_window=\u00bbon\u00bb button_text=\u00bbaccess video content\u00bb button_alignment=\u00bbleft\u00bb _builder_version=\u00bb4.16″ custom_padding=\u00bb10px|60px|10px|60px|true|true\u00bb custom_padding_tablet=\u00bb\u00bb custom_padding_phone=\u00bb|30px||30px||true\u00bb custom_padding_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb button_text_size__hover_enabled=\u00bboff\u00bb button_one_text_size__hover_enabled=\u00bboff\u00bb button_two_text_size__hover_enabled=\u00bboff\u00bb button_text_color__hover_enabled=\u00bboff\u00bb button_one_text_color__hover_enabled=\u00bboff\u00bb button_two_text_color__hover_enabled=\u00bboff\u00bb button_border_width__hover_enabled=\u00bboff\u00bb button_one_border_width__hover_enabled=\u00bboff\u00bb button_two_border_width__hover_enabled=\u00bboff\u00bb button_border_color__hover_enabled=\u00bboff\u00bb button_one_border_color__hover_enabled=\u00bboff\u00bb button_two_border_color__hover_enabled=\u00bboff\u00bb button_border_radius__hover_enabled=\u00bboff\u00bb button_one_border_radius__hover_enabled=\u00bboff\u00bb button_two_border_radius__hover_enabled=\u00bboff\u00bb button_letter_spacing__hover_enabled=\u00bboff\u00bb button_one_letter_spacing__hover_enabled=\u00bboff\u00bb button_two_letter_spacing__hover_enabled=\u00bboff\u00bb button_bg_color__hover_enabled=\u00bboff\u00bb button_one_bg_color__hover_enabled=\u00bboff\u00bb button_two_bg_color__hover_enabled=\u00bboff\u00bb][\/et_pb_button][\/et_pb_column][\/et_pb_row][et_pb_row module_class=\u00bb et_pb_row_fullwidth\u00bb _builder_version=\u00bb4.16″ width=\u00bb89%\u00bb width_tablet=\u00bb80%\u00bb width_last_edited=\u00bbon|desktop\u00bb max_width=\u00bb89%\u00bb max_width_tablet=\u00bb80%\u00bb max_width_last_edited=\u00bbon|desktop\u00bb make_fullwidth=\u00bbon\u00bb global_colors_info=\u00bb{}\u00bb width_phone=\u00bb80%\u00bb max_width_phone=\u00bb80%\u00bb][et_pb_column type=\u00bb4_4″ _builder_version=\u00bb4.16″ custom_padding=\u00bb|||\u00bb global_colors_info=\u00bb{}\u00bb custom_padding__hover=\u00bb|||\u00bb][et_pb_text _builder_version=\u00bb4.23.1″ text_font=\u00bb||||||||\u00bb text_line_height=\u00bb1.3em\u00bb header_font=\u00bb||||||||\u00bb header_2_font=\u00bb|700|||||||\u00bb header_2_text_color=\u00bb#0a2d31″ header_2_font_size=\u00bb33px\u00bb hover_enabled=\u00bb0″ text_font_size_tablet=\u00bb\u00bb text_font_size_phone=\u00bb18px\u00bb text_font_size_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb module_id=\u00bb4-2″ sticky_enabled=\u00bb0″]<\/p>\n These methods are based on the discharge of intracellular enzymes to the growth medium when the bacteria lyse<\/strong>. This kind of method requires identifying easy-to-detect intracellular enzymes, such as adenylate kinase and adenosine 5\u2019-triphosphate, \u03b2-galactosidase and \u03b2- glucuroronidase, for which chromogenic or fluorogenic substrates are available (Guzman et al. 2009, Salter et al., 2010, Muniesa et al., 2018)<\/p>\n At present this procedure is only applicable to liquid suspension<\/strong> and hence for presence\/absence<\/strong> determination. However, as explained in the previous section, accurate quantal methods such as most probable number (MPN)<\/strong>, can be used.<\/p>\n [\/et_pb_text][et_pb_text _builder_version=\u00bb4.23.1″ text_font=\u00bb||||||||\u00bb text_line_height=\u00bb1.3em\u00bb header_font=\u00bb||||||||\u00bb header_2_font=\u00bb|700|||||||\u00bb header_2_text_color=\u00bb#0a2d31″ header_2_font_size=\u00bb33px\u00bb hover_enabled=\u00bb0″ text_font_size_tablet=\u00bb\u00bb text_font_size_phone=\u00bb18px\u00bb text_font_size_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb module_id=\u00bb4-3″ sticky_enabled=\u00bb0″]<\/p>\n Based on these principles, the following standardized methods are available for bacteriophages proposed or being used as indicators. The key feature of standardized methods is the host bacteria selected for the method.<\/p>\n [\/et_pb_text][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_font_size=\u00bb18px\u00bb text_line_height=\u00bb1.2em\u00bb background_color=\u00bb#f0f1ec\u00bb custom_padding=\u00bb50px|50px|50px|50px|true|true\u00bb custom_padding_tablet=\u00bb\u00bb custom_padding_phone=\u00bb30px|30px|30px|30px|true|true\u00bb custom_padding_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n International Standardization Organization.<\/strong> Water Quality\u2014Detection and Enumeration of Bacteriophages\u2014Part 1: Enumeration of F-specific RNA Bacteriophages; ISO-10705-1; International Standardization Organization: Geneva, Switzerland, 1995.<\/em><\/p>\n [\/et_pb_text][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_font_size=\u00bb18px\u00bb text_line_height=\u00bb1.2em\u00bb custom_padding=\u00bb|50px||50px|true|true\u00bb custom_padding_tablet=\u00bb\u00bb custom_padding_phone=\u00bb|0px||0px||true\u00bb custom_padding_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n International Standardization Organization.<\/strong> Water Quality\u2014Detection and Enumeration of Bacteriophages\u2014Part 2: Detection and Enumeration of Somatic Coliphages; ISO-10705-2; International Standardization Organization: Geneva, Switzerland, 2000.<\/em><\/p>\n [\/et_pb_text][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_font_size=\u00bb18px\u00bb text_line_height=\u00bb1.2em\u00bb background_color=\u00bb#f0f1ec\u00bb custom_padding=\u00bb50px|50px|50px|50px|true|true\u00bb custom_padding_tablet=\u00bb\u00bb custom_padding_phone=\u00bb30px|30px|30px|30px|true|true\u00bb custom_padding_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n USEPA Office of Water.<\/strong> Method 1601: Male-specific (F+) and Somatic Coliphage in Water by Two-step Enrichment Procedure; EPA 821-R-01-030; USEPA Office of Water: Washington, DC, USA, 2001.<\/em><\/p>\n [\/et_pb_text][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_font_size=\u00bb18px\u00bb text_line_height=\u00bb1.2em\u00bb custom_padding=\u00bb|50px||50px|true|true\u00bb custom_padding_tablet=\u00bb\u00bb custom_padding_phone=\u00bb|0px||0px||true\u00bb custom_padding_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n USEPA Office of Water.<\/strong> Method 1601: Male-specific (F+) and Somatic Coliphage in Water by Two-step Enrichment Procedure; EPA 821-R-01-030; USEPA Office of Water: Washington, DC, USA, 2001.<\/em><\/p>\n [\/et_pb_text][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_font_size=\u00bb18px\u00bb text_line_height=\u00bb1.2em\u00bb background_color=\u00bb#f0f1ec\u00bb custom_padding=\u00bb50px|50px|50px|50px|true|true\u00bb custom_padding_tablet=\u00bb\u00bb custom_padding_phone=\u00bb30px|30px|30px|30px|true|true\u00bb custom_padding_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n USEPA Office of Water<\/strong>. Method 1602: Male-specific (F+) and Somatic Coliphage in Water by Single Agar Layer (SAL) Procedure; EPA 821-R-01-029; USEPA Office of Water: Washington, DC, USA, 2001.<\/em><\/p>\n [\/et_pb_text][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_font_size=\u00bb18px\u00bb text_line_height=\u00bb1.2em\u00bb custom_margin=\u00bb||0px|\u00bb custom_padding=\u00bb0px|50px|0px|50px|true|true\u00bb custom_padding_tablet=\u00bb\u00bb custom_padding_phone=\u00bb|||0px\u00bb custom_padding_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n Rice, E.W.,Baird, R.B., Eaton, A.D. and Clesceri, L.S. Standard Methods for the Examination of Water and Wastewater<\/strong>; American Public Health Association, American Water Works Association, Water Environment Federation. Washington, DC, USA, 2012<\/em>.<\/p>\n \u00a0<\/em><\/p>\n [\/et_pb_text][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_font_size=\u00bb18px\u00bb text_line_height=\u00bb1.2em\u00bb background_color=\u00bb#f0f1ec\u00bb custom_padding=\u00bb50px|50px|50px|50px|true|true\u00bb custom_padding_tablet=\u00bb\u00bb custom_padding_phone=\u00bb30px|30px|30px|30px|true|true\u00bb custom_padding_last_edited=\u00bbon|phone\u00bb locked=\u00bboff\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n International Standardization Organization.<\/strong> Water Quality\u2014Detection and Enumeration of Bacteriophages-Part 4: Detection and enumeration of bacteriophages infecting Bacteroidesfragilis; ISO-10705-2 International Standardization Organization, Geneva, Switzerland, 2001<\/em><\/p>\n [\/et_pb_text][et_pb_divider _builder_version=\u00bb4.16″ global_colors_info=\u00bb{}\u00bb][\/et_pb_divider][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_line_height=\u00bb1.3em\u00bb header_font=\u00bb||||||||\u00bb header_2_font=\u00bb|700|||||||\u00bb header_2_text_color=\u00bb#0a2d31″ header_2_font_size=\u00bb33px\u00bb text_font_size_tablet=\u00bb\u00bb text_font_size_phone=\u00bb18px\u00bb text_font_size_last_edited=\u00bbon|phone\u00bb locked=\u00bboff\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n Some emerging detection methods are based on the detection of an enzyme released by cell lysis into the liquid matrix where phage infection of a host cell occurs. These methods appear to be simple, fast, cost-effective and as accessible as user-friendly kits.<\/p>\n [\/et_pb_text][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_font_size=\u00bb28px\u00bb text_line_height=\u00bb1.2em\u00bb background_color=\u00bb#f0f1ec\u00bb text_orientation=\u00bbcenter\u00bb custom_margin=\u00bb|||||true\u00bb custom_padding=\u00bb50px|50px|70px|50px||true\u00bb custom_padding_tablet=\u00bb\u00bb custom_padding_phone=\u00bb50px|30px|50px|30px|true|true\u00bb custom_padding_last_edited=\u00bbon|phone\u00bb text_font_size_tablet=\u00bb\u00bb text_font_size_phone=\u00bb21px\u00bb text_font_size_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n BLUEPHAGE is a method based on this principle for which engineered host cells have been designed to increase the specificity, sensitivity and speed of the procedure<\/strong>.<\/p>\n [\/et_pb_text][\/et_pb_column][\/et_pb_row][et_pb_row module_class=\u00bb et_pb_row_fullwidth\u00bb _builder_version=\u00bb4.16″ width=\u00bb89%\u00bb width_tablet=\u00bb80%\u00bb width_last_edited=\u00bbon|desktop\u00bb max_width=\u00bb89%\u00bb max_width_tablet=\u00bb80%\u00bb max_width_last_edited=\u00bbon|desktop\u00bb custom_padding=\u00bb31.7833px|0px|4px|0px|false|false\u00bb make_fullwidth=\u00bbon\u00bb global_colors_info=\u00bb{}\u00bb width_phone=\u00bb80%\u00bb max_width_phone=\u00bb80%\u00bb][et_pb_column type=\u00bb4_4″ _builder_version=\u00bb4.16″ custom_padding=\u00bb|||\u00bb global_colors_info=\u00bb{}\u00bb custom_padding__hover=\u00bb|||\u00bb][et_pb_divider divider_weight=\u00bb2px\u00bb _builder_version=\u00bb4.16″ max_width=\u00bb20%\u00bb global_colors_info=\u00bb{}\u00bb][\/et_pb_divider][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb|700|||||||\u00bb text_text_color=\u00bb#256168″ text_font_size=\u00bb33px\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n References<\/p>\n [\/et_pb_text][et_pb_text _builder_version=\u00bb4.16″ text_font=\u00bb||||||||\u00bb text_font_size=\u00bb15px\u00bb text_line_height=\u00bb1.3em\u00bb background_color=\u00bb#eaeddb\u00bb custom_padding=\u00bb50px|50px|50px|50px|true|true\u00bb custom_padding_tablet=\u00bb\u00bb custom_padding_phone=\u00bb|30px||30px||true\u00bb custom_padding_last_edited=\u00bbon|phone\u00bb text_font_size_tablet=\u00bb\u00bb text_font_size_phone=\u00bb12px\u00bb text_font_size_last_edited=\u00bbon|phone\u00bb global_colors_info=\u00bb{}\u00bb]<\/p>\n Adams, M.H. (1959).<\/em> Bacteriophages<\/strong>. Interscience Publishers Inc. New York.<\/p>\n Guzm\u00e1n Luna, C., Cost\u00e1n-Longares, A., Lucena, F. and Jofre, J.<\/em> Detection of somatic coliphages through a bioluminescence assay measuring phage mediated release of adenylate kinase and adenosine 5\u2019-triphosphate<\/strong>. J. Virol. Methods 2009, 161, 107\u2013113.<\/p>\n Salter, R.S. and Durbin, G.W.<\/em> Modified USEPA method 1601 to indicate viral contamination of groundwater<\/strong>. J. Am. Water Works Assoc. 2012, 104, 480\u2013488.<\/p>\n Muniesa, M., Ballest\u00e9, E., Imamovic, L., Pascula-Benito, M., Toribio-Avedillo, D., Lucena, F.and and Jofre, J. 2018<\/em>. Bluephage: A rapid method for the detection of somatic coliphages used as indicators of fecal pollution in water<\/strong>. Water Research 128: 10-19.<\/p>\n [\/et_pb_text][\/et_pb_column][\/et_pb_row][\/et_pb_section][et_pb_section fb_built=\u00bb1″ _builder_version=\u00bb3.12.1″ background_color=\u00bb#256168″ custom_padding=\u00bb33px|0px|0|0px|false|false\u00bb global_module=\u00bb97″ saved_tabs=\u00bball\u00bb global_colors_info=\u00bb{}\u00bb][et_pb_row module_class=\u00bb et_pb_row_fullwidth\u00bb _builder_version=\u00bb4.16″ width=\u00bb89%\u00bb width_tablet=\u00bb80%\u00bb width_last_edited=\u00bbon|desktop\u00bb max_width=\u00bb89%\u00bb max_width_tablet=\u00bb80%\u00bb max_width_last_edited=\u00bbon|desktop\u00bb module_alignment=\u00bbcenter\u00bb custom_margin=\u00bb0px|||\u00bb custom_padding=\u00bb0px||0||false|false\u00bb make_fullwidth=\u00bbon\u00bb global_colors_info=\u00bb{}\u00bb column_structure=\u00bb1_3,1_3,1_3″ width_phone=\u00bb80%\u00bb max_width_phone=\u00bb80%\u00bb][et_pb_column type=\u00bb1_3″ _builder_version=\u00bb4.16″ custom_padding=\u00bb||30px|\u00bb global_colors_info=\u00bb{}\u00bb custom_padding__hover=\u00bb|||\u00bb][et_pb_image src=\u00bbhttps:\/\/coliphages.ixole.es\/wp-content\/uploads\/2019\/05\/logo-coliphages.png\u00bb show_bottom_space=\u00bboff\u00bb align=\u00bbright\u00bb align_tablet=\u00bbcenter\u00bb align_last_edited=\u00bbon|desktop\u00bb _builder_version=\u00bb4.16″ custom_margin=\u00bb0px|||\u00bb custom_padding=\u00bb|30px|30px|\u00bb global_colors_info=\u00bb{}\u00bb align_phone=\u00bbcenter\u00bb][\/et_pb_image][\/et_pb_column][et_pb_column type=\u00bb1_3″ _builder_version=\u00bb4.16″ custom_padding=\u00bb|||\u00bb global_colors_info=\u00bb{}\u00bb custom_padding__hover=\u00bb|||\u00bb][et_pb_image src=\u00bbhttps:\/\/coliphages.ixole.es\/wp-content\/uploads\/2018\/09\/ub-mars.png\u00bb align_tablet=\u00bbcenter\u00bb align_last_edited=\u00bbon|desktop\u00bb _builder_version=\u00bb4.16″ custom_margin=\u00bb30px|||30px\u00bb global_colors_info=\u00bb{}\u00bb align_phone=\u00bbcenter\u00bb][\/et_pb_image][\/et_pb_column][et_pb_column type=\u00bb1_3″ _builder_version=\u00bb4.16″ custom_padding=\u00bb|||\u00bb global_colors_info=\u00bb{}\u00bb custom_padding__hover=\u00bb|||\u00bb][et_pb_sidebar area=\u00bbsidebar-4″ disabled_on=\u00bbon|on|off\u00bb _builder_version=\u00bb4.16″ header_font=\u00bb|700|||||||\u00bb header_text_color=\u00bb#dce09a\u00bb header_font_size=\u00bb16px\u00bb background_layout=\u00bbdark\u00bb custom_margin=\u00bb40px|||30px\u00bb global_colors_info=\u00bb{}\u00bb][\/et_pb_sidebar][\/et_pb_column][\/et_pb_row][\/et_pb_section]<\/p>\n","protected":false},"excerpt":{"rendered":" Detection and enumeration methods 4.1 Methods based on direct observation of host cell lysis4.2 Methods based on the determination of molecules released by cell lysis4.3 Specific methodsBefore starting to look at detection and enumeration, we should perhaps talk a little bit about sampling and conservation of the samples. When taking a sample the ideal situation […]<\/p>\n","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_et_pb_use_builder":"on","_et_pb_old_content":"","_et_gb_content_width":"","_monsterinsights_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0,"footnotes":""},"class_list":["post-209","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/coliphages.ixole.es\/index.php\/wp-json\/wp\/v2\/pages\/209","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/coliphages.ixole.es\/index.php\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/coliphages.ixole.es\/index.php\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/coliphages.ixole.es\/index.php\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/coliphages.ixole.es\/index.php\/wp-json\/wp\/v2\/comments?post=209"}],"version-history":[{"count":67,"href":"https:\/\/coliphages.ixole.es\/index.php\/wp-json\/wp\/v2\/pages\/209\/revisions"}],"predecessor-version":[{"id":952,"href":"https:\/\/coliphages.ixole.es\/index.php\/wp-json\/wp\/v2\/pages\/209\/revisions\/952"}],"wp:attachment":[{"href":"https:\/\/coliphages.ixole.es\/index.php\/wp-json\/wp\/v2\/media?parent=209"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}
4.2<\/strong> Methods based on the determination of molecules released by cell lysis<\/a>
4.3<\/strong> Specific methods<\/a><\/p>\n4.1 Methods based on direct observation of host cell lysis<\/h2>\n
4.2 Methods based on the determination of molecules released by cell lysis<\/h2>\n
4.3 Specific methods<\/h2>\n
Standardized methods<\/em><\/h3>\n
Emerging methods<\/em><\/h3>\n