Biotechnology of Microorganisms

Diversity, Improvement, and Application of Microbes for Food Processing, Healthcare, Environmental Safety, and Agriculture

Jeyabalan Sangeetha, Devarajan Thangadurai, Somboon Tanasupawat, Pradnya Pralhad Kanekar, Jeyabalan Sangeetha, Devarajan Thangadurai, Somboon Tanasupawat, Pradnya Pralhad Kanekar

• Publisher: Apple Academic Press
• Year: 2019
• ISBN:
  9780429784019
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Science General
• Index: Biological Sciences

Microbial biotechnology is an important contributor to global business, especially in agriculture, the environment, healthcare, and the medical, food, and chemical industries. This volume provides an exciting interdisciplinary journey through the rapidly changing backdrop of invention in microbial biotechnology, covering a range of topics, including microbial properties and characterization, cultivation and production strategies, and applications in healthcare, bioremediation, nanotechnology, and more.

Key features:

• 
  

• Explains the diverse aspects of and strategies for cultivation of microbial species

• 
  
  

  
  

• Describes biodiversity and biotechnology of microbes

• 
  
  

  
  

• Provides an understanding of microorganisms in bioremediation of pollutants

• 
  
  

  
  

• Explores various applications of microbes in agriculture, food, health, industry, and the environment

• 
  
  

  
  

• Considers production issues and applications of microbial secondary metabolites

• 
  
  

  
  

• Underscores the importance of integrating genomics of microorganisms in ecological restoration of contaminated environments

CHAPTER 1

COMPARATIVE STUDIES ON CFU DETERMINATION BY PURE AND/OR MIXED CULTURE CULTIVATIONS

SUBIR KUMAR NANDY

Technical University of Denmark, Søltofts Plads, 2800 Kgs, Lyngby, Denmark

1.1 INTRODUCTION

Existing methodologies used in the determination of metabolic active cell count (MACC) are either time consuming, laborious, error-prone or expensive. Studies of any culture require an easy and fast methodology for quantification of MACC of different types of microorganisms. Thus, it is necessary to combine all probable methodology for quantification of MACC in the biological studies. Further, such a platform should be required to compare rapid and precise methodologies for viable cell quantification so that it could be easier to evaluate the dynamic response of any culture. Thus, the overall objective of this chapter is to develop one platform for a quantitative method to resolve MACC in biological cultures. Furthermore, these quantification processes are crucial in the pharmaceutical and food industries for epidemiologic investigation.

The study of the above phenomenon requires quantification of MACC in pure and mixed cultures. Mixed cultures are usually used in several different processes involving bioremediation and food processing. Quantification of MACC of mixed cultures is monotonous. Quantification of more than one culture may be helpful in characterizing contamination in fermentation processes. Existing methodologies used in the determination of MACC are either time consuming or expensive.

Thus, it is necessary to generate a process to quantify MACC in mixed cultures. The method should not only yield a total cell count of all the microorganisms, but also yield the MACC of individual organism present in the culture having more than one microorganism.

1.2 METHODS FOR VIABLE CELL COUNT

1.2.1 PLATE COUNT TECHNIQUES

This normal method is used to estimate cell culturability. Here, agar plates are used to obtain CFU (colony forming units).

1.2.1.1 SPREAD PLATE TECHNIQUE

The total number of bacteria in the experimental flask can be quantified by using spread plate (SP) techniques. In this process sample is diluted several times depending on the growth of organisms and a fraction is transferred to an agar plate. The bacteria are evenly spread over the surface of agar by SP method. The procedure is carried out in a laminar hood to minimize contamination. The colonies are grown overnight in the incubator with specific temperature depending on the type of strain to be quantified. All the colonies are counted, and total bacteria are calculated depending on dilution. Finally, the total number of bacteria is determined based on a serial dilution and plating them serially. The most important advantage of this process is that only metabolically active cells grow on the agar (Kirkpatrick et al., 2001).

The precaution of this method is that all the plates and tips should be autoclaved for less chance of contamination. Sterilized plates are also available now, and that saves the time for sterilization. The bent glass rod is used to spread the culture over the plates. The glass rod should be decontaminated by dipping into ethanol and holding in Bunsen burner to avoid contamination. Finally, the plate should be counted when colonies are in the range of 40–200. The total number of colonies can be calculated by multiplying the average colonies with the dilution factor in terms of CFU/ml. The major advantage of the method is its simplicity, with the disadvantage being that the method is a time-consuming process.

1.2.1.2 STREAK PLATE TECHNIQUE

In this method, the microorganism is streaked on the agar using sterilized loop. This method is used to separate pure colony from a mixed culture or from contamination and cannot be used for determining bacterial count. The colony separation procedure is also depended on the dilution of the broth.

1.2.2 STAINING PROCESSES

1.2.2.1 DYE EXCLUSION METHOD

One of the widely used techniques is the dye exclusion test. This method is based on specific dyes such as methylene blue, trypan blue and acridine orange (Sampson et al., 1924). This procedure works where dead cells are stained, but active metabolic cells remain unstained when exposed to the dye. The advantage of this process is that this assay is easy and fast.

This process is not an accurate method (error-prone technique). Cells are not stained while culture was stored for a long time at low temperatures (Hauschka et al., 1959). Other disadvantages are (a) effective concentration of trypan blue is based on the cell suspension; and (b) maintaining the exact condition of sample preparation was difficult (Pappenheimer et al., 1917).

1.2.2.2 GRAM POSITIVE AND GRAM NEGATIVE STAINING

1.2.2.2.1 Slide Gram Stain

Gram staining method is not suggested for the whole broth quantification procedure. This method is used to separate gram positive and gram negative bacteria (Gram, 1884) very well. The exact mechanism of the gram stain is not clearly understood and due to the errors in staining the procedure is not effective (Beveridge and Davies, 1983; Davies et al., 1983). The disadvantage of this process is that it cannot detect CFU at low concentrations (<1000 live cells).

1.2.2.2.2 Filter Gram Stain

Microfiltration has become a popular procedure for the concentration and enumeration of bacteria. This is a rapid and a sensitive procedure than the normal slide gram stain method to differentiate bacteria utilizing a polycarbonate membrane filter, crystal violet, iodine, 95% ethanol, and 6% carbol fuchsin that can be completed in 60 to 90 s. This method is useful to detect less number of bacteria (∼ 100 cells) but requires modification in the protocol for specific systems (Romero et al., 1988).

1.2.3 MICROSCOPIC PROCEDURE

1.2.3.1 GENERAL APPROACH

A microscopic approach can be used to calculate live cells directly within a very short time, but this approach is not accurate. The culture was stained and fixed on slides to quantify under a microscope (Zhang and Shen, 2006). Few researchers used hemocytometer to quantify the microorganisms.

1.2.3.2 IMMUNOFLUORESCENCE AND EPIFLUORESCENCE ASSAY

These assays are used for labeling antibodies and antigens with fluorescent dyes (Zhang and Shen, 2006). Direct epifluorescence counting was also a suitable method for enumeration of total bacteria in environmental samples (Kepner et al., 1994). Dunne et al. (1987) use of immunofluorescence have been outmoded by the development of recombinant proteins containing fluorescent protein domains, e.g., a green fluorescent protein (GFP).

1.2.3.3 CONFOCAL SCANNING LASER MICROSCOPE ASSAY

Confocal scanning laser microscope (CSLM) is a technique for obtaining high-resolution optical images. Depending on the fluorescence properties of the used dyes, there is a subtle improvement in lateral resolution compared to conventional microscopes. This is a fast method compared to other available techniques to detect the active metabolic cells from heat-killed bacteria than standard SP method. This CSLM method is specially used in the dairy industry to check microbial or contamination load in milk (Pettipher et al., 1980). This method also quantifies bacteria rapidly by image analysis (Caldwell et al., 1992).

1.2.3.4 FLUORESCENCE MICROSCOPIC METHOD

In this process cells inoculated on the chamber, slides were fixed in methanol at −20°C for 10 min for cyclin B1 detection. The cells for microtubule detection were fixed with a 10% formaldehyde solution containing 0.5% Triton X−100, 1 mM MgCl₂ at room temperature for 10 min and further fixed with the same solution without Triton X−100 for 5 min. The fixed cells were washed with cold PBS and incubated overnight with a monoclonal anti-cyclin B1 (Pharmingen) or anti-β-tubulin antibody (Sigma) in 1% BSA/PBS at 4°C. They were washed and incubated with a FITC-conjugated anti-mouse IgG antibody (Pharmingen) in 1% BSA/PBS in the dark at room temperature for 1 h. The cells were then washed and stained with 0.2 µg/ml Hoechst 33258 (bis-benzimide trihydrochloride, Sigma) at room temperature for 10 min. The slide was washed and mounted in glycerol. The cells were viewed on a fluorescence microscope using epi-illumination (BX60, Olympus Optics, Tokyo, Japan) and photographed on Tmax film (Kodak, ASA 400). This assay is also a rapid process for direct assessment of cell viability (Kepner and Pratt, 1994). Very few researchers were used LIVE/DEAD BacLight Viability Kit (Molecular Probes Inc., Eugene, OR) to differentiate live/dead bacteria based on plasma membrane permeability (Virta, 1998).

1.2.4 DROP PLATE COUNT METHOD

Metabolically active cells present in a known volume can be determined by the drop plate (DP) method. Though the DP method is not optimized, recent studies have shown some advantages over traditional SP method. Colony counting in DP method is very accurate and faster but depending on various parameters such as dilutions which may vary from laboratory to laboratory or technician to technician. DP method is a quick and easy process where drops can be dispensed onto an agar plate compared to SP technique where the sample volume spread on the agar. Few researchers use 10-fold dilutions, others use two-fold, or few laboratories use a total volume of 0.1 ml on a plate, others plate 0.2 ml, but all these factors use while practicing the DP method.

This assay is used for an active metabolic cell which is faster than SP technique (Herigstad et al., 2001). In this assay, the culture drops on agar in a little volume, and it will absorb quickly. Several researchers want to demonstrate this assay for bacterial counts (Herigstad et al., 2001). In this method, the small drops are separated on agar, and after incubation of the plates, colonies within the drops are counted, and finally, overall bacteria count based on the flask volume. This method is not useful for swarming type bacteria such as Proteus mirabilis and Proteus vulgaris. This method is a labor-intensive procedure.

1.2.5 INSTRUMENTATION PROCESSES

PCR and Flow Cytometry (FCM) can also yield valuable information regarding viability (Davey and Kell, 1996; Decre et al., 1998). FCM is a means of measuring certain physical and chemical characteristics of cells or particles as they pass in a fluid stream by a beam of laser light. The term “flow cytometry” derives from the measurement (meter) of single cells (cyto) as they flow past a series of detectors. Flow sorting extends FCM by using electrical or mechanical means to divert and collect cells with one or more measured characteristics falling within a range or ranges of values set by the user. The major applications of FCM include the analysis of cell cycle, apoptosis, necrosis, multicolor analysis, cell sorting, functional analysis, and stem cell analysis. Oravcova et al. (2008) show that real-time P...