Staining of bacteria by fluorescently labeled bacteriophages

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STAINING OF BACTERIA BY FLUORESCENTLY LABELED
BACTERIOPHAGES
by M.A.Khan, D.Sharma, D.Gahloth, B.M.J Pereira
Department of Biotechnology Indian Institute of
Technology Roorkee (IITR) Roorkee-247667,
Uttaranchal, INDIA e-mailmkhanfbs_at_iitr.ernet.in
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Contents

• Basic Introduction
• Fluorescently labeled phages
• Introduction
• Methodology development
• Host range
• Detection of activated sludge bacteria
• Conclusions

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Basic Introduction

• Bacteriophages are viruses of bacteria.
• Bacteriophages are extremely abundant and active
  member of the biological wastewater treatment
  system.
• Several bacterial species involved in biological
  wastewater treatment have a significant impact on
  process efficiency so the interaction of bacteria
  and their predators, such as phages, is of
  interest.
• Bacteriophages are environmentally important both
  in controlling bacterial numbers and in
  facilitating bacterial gene transfer.

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What are Fluorescentlylabeled bacteriophages?

• Several fluorescent dyes can stain bacteriophage
  nucleic acid which can be easily visualized by
  epifluorescence microscope.
• DAPI
• Acridine orange
• FITC (flurescein-5-isothiocyanate)
• SYBR

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Significance of Fluorescently labeled
bacteriophage

• They can be used to identify and quantify the
  specific strains of bacteria in aquatic samples.
• They can be applied to activated sludge system or
  any other natural environment where diverse kinds
  of bacteria exist.

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Objectives

• To develop methodology to label bacteriophages
  isolated
• from activated sludge by fluorescent dyes.
• To evaluate the feasibility of the fluorescently
  labeled
• Phages to examine host range on several host
• bacteria.
• To evaluate the scope of for the detection of
  sensitive
• bacterial cells within mixed culture.
• (activated sludge samples)

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Bacteriophage bacteria isolation method
Activated sludge mixed liquor
Elution of sludge particles by 10 beef extract
solution
Incubation
Plating method
Picking-up a colony streaking plating method
Centrifugation and filtration
Filtrate host bacteria
Purified isolate
Plaque formation test (observation of plaques)
Picking individual plaques and purification
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Prerequisite of preparing Fluorescently labeled
Phages

• Selection of the phage-host system
• Preparation of high titer phage stock
• Purification of phage stock

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P30- ?P30 phage-host system
characterization

• P30 host is a Gram-ve rods
• identified as Serratia marcescens
• Plaque formation time was rapid and
• reproducible
• Plaque size were big(2-3 mm)
• ?P30 is a DNA phage

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Optimum growth conditions for (?P30, ?P35, ?P37
phages)

• Optimum time Optimum
  Lysis
• for adding phage temperature
  time
• ?P30 2 hrs 30?C
  67 hrs
• 
  (complete)
• ?P35 1 hrs 30?C
  34 hrs
• 
  (partial)
• ?P37 2 hrs 30?C
  34 hrs
• 
  (partial)

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Fluorescent labeling of phage
Host cells phage fluorescent dye
Treat lysate with DNase to release labeled
phages from the mixture PEG1M NaCl
Purification of labeled phage by
Ultra-filtration
Mixing with its host and visualize under
epifluroscent microscope
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DAPI stained P30 host cells (control) without
phage
Purified DAPI labeled ?P30 phage particles after
Ultra filtration
10 µm
10 µm
Epifluroscent image, ?1000 Magnification
Epifluroscent image, ?1000 Magnification
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Application of DAPI-labeled phage

• Detection of the host bacterial
• cells in pure culture
• Cross reactivity with
• other isolates (host range)

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Purified phage particles infecting its host (pure
culture) after mixing
I
II
III
IV
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?P30 cross infectivity with different activated
sludge isolates
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Application of DAPI-labeled phage to activated
sludge samples

• Activated sludge samples host cells
• (with or without sonication)
• Activated sludge samples no host cells
• (with or without sonication)

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P30 host Activated sludge sample labeled phage
stock
Bright field image,?1000 Magnification
Epifluroscent image, ?1000 Magnification
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P30 host Activated sludge sample DAPI
labeled phage (100? dilution)
Epifluroscent image, ?1000 Magnification
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DAPI-labeled phage application to activated
sludge samples(without added host cells)
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Conditions of using FLP in activated sludge

• Viable host cells must be present in the
• samples
• Activated sludge samples should be
• well dispersed
• Fluorescently labeled phage particles
• should be well purified
• (free from residual dyes)

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Conclusions

• Phage ?P30 DNA was labeled successfully by DAPI
  (1mg/l) and Acridine orange (50mg/l).
• DAPI labeled ?P30 phage particles were able to
  detect their unlabeled host cells in their pure
  culture.
• The bacteriophage particles did not lose their
  infectivity even after labeling their nucleic
  acid by DAPI (conc1mg/l).

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Conclusions

• DAPI labeled phage particles can detect its host
  cells in the activated sludge samples.
• Cross reactivity of the labeled phage with other
  activated sludge isolates shows that it was
  specific for its host.

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Thanks