Microbiology Project Topics

Analysis of Various Microbial Staining Techniques

Analysis of Various Microbial Staining Techniques

Analysis of Various Microbial Staining Techniques

Chapter One


  • To study the morphology of the microorganisms.
  • To practice the staining methods used to study microbial morphology.



Floc formation and settling are the processes required after the goal of organic waste removal is completed. The evaluation of settleability and flocculation following the completion of the organic waste removal process can be determined at various scales. At the wastewater treatment plant scale, parameters such as sludge volume index (SVI), mixed liquor suspended solid (MLSS), sludge retention time (SRT) and quantitative assays of organic waste matter content for the influent and effluent waters are important. At the floc scale, the composition of the microbial community, physicochemical parameters and microscopic features are the focus. Although the internal architecture of the floc can be revealed at the nanometer scale using transmission electron microscopy (TEM), it will be listed in the floc scale evaluation section in this thesis. Finally, at the sub-floc level EPS composition and the distribution of EPS constituents become significant. Understanding the distribution of EPS constituents at various depths of floc, is the focus of this research.

 Evaluation at the wastewater treatment plant scale

Floc formation and settling in activated sludge can be assessed using two measurements, namely the MLSS and SVI. MLSS and SVI are routine tests at the macro scale to assess performance of the reactor. MLSS is a measure of suspended solids in the sample. Although flocculation is not greatly affected by the concentration of suspended solids, there are reports in the literature describing the negative influence of high MLSS on effluent quality 24,158. MLSS is a measure of mixed liquor suspended solids and this measure includes the total weight of microorganisms, EPS, organic waste, suspended waste and any other particulate in wastewater 64.

SVI is a measure of sludge settleability. SVI is defined as the volume in millimeters occupied by one gram of suspension after 30 min 5. It indirectly measures morphology of flocs, and is a physical characteristic of activated sludge 99,137. SVI is measured at the macro level and it tracks the settling of a sludge sample rather than the settling of one single particle. SVI needs to be measured as a function of MLSS. The MLSS consideration is only accurate for sludge samples up to 4000 mg/L, MLSS values higher than 4000 mg/L would introduce errors in the SVI measurement 40. This makes SVI theoretically not supported, but it is a useful assessment of process control.

Furthermore, since it is simple, inexpensive, and fast this test is still considered to be a routine test 40,48.

SRT is not a test but an operational parameter that states, how long the sludge has been retained; in other words, it is the cell residence time in a reactor. SRT may influence many other characteristics of activated sludge, including: hydrophobicity, surface charge, surface irregularity and EPS 98,99.

In addition to SRT, other carefully controlled operational parameters are essential to microbial well-being. Microbial cells could be considered an ongoing progress of evolution and as a result, they demand certain optimized conditions for their survival.

These conditions include: pH, temperature, food to microorganism ratio and ratio of different nutrients 1,10,80,102. The above conditions are all necessary for the survival of microorganisms in their niche. In WWTP, the above conditions are not easy to maintain optimally at all times due to parameters such as variability of influent water or weather conditions.

When the above conditions are not optimized, the microbial community may change 17,31. Changes in the community may cause inefficiencies in reactor performance along with changes in settleability and/or formation of solid/liquid interfaces 20,82,119.

Formation of solid/liquid interfaces is dependent on the stabilization of physicochemical properties in a floc 92,98,104.

Evaluation at the floc scale

A floc is a complicated structure in which microscopic features such as the population of floc formers versus filamentous microorganisms may influence the drag force and, therefore, affect settleability of flocs. Furthermore physicochemical properties such as surface charge and hydrophobicity may also play a role in settleability as discussed earlier. Finally the internal architecture, i.e. compactness of the floc structure may influence the quantity of bound water and free water and impact settleability 74.

Mixed microbial flocs are thick, dense structures and have a complicated architecture, such that various microscopic techniques are utilized to examine the floc structure. Conventional optical microscopy (COM) relies on visible light and due to the random scattering of light at the floc surface, COM may not be able to reveal the internal structure of the flocs 38,85. Reliability of confocal laser scanning microscopy (CLSM) data is partially dependent upon homogenous diffusion of fluorescent probes, section thickness and specific binding of the fluorescent probes to the targeted epitope 90,165.

TEM includes sample preparation steps with harsh chemicals such glutaraldehyde and osmium tetroxide for chemical fixation followed by treatment with acetone for dehydration 22. The processing causes protein reconfiguration, lipid extraction and finally nucleic acid condensation 78. Each of the above mentioned microscopic methods have built in limitations, biases and artifacts. Therefore, to have results with minimal bias, multiple microscopy tools must be used in conjunction with one another. Use of multiple microscopic tools was first defined by Leppard (1979) as correlative microscopy and successfully applied in the field of wastewater treatment to flocs by Liss et al. (1997).




  • Culture plates of Escherichia coli, Staphylococcus aureus and old cultures of Bacillus subtilis spizizenni.
  • Inoculating loop
  • Bunsen burner
  • Sterile water
  • Clean slides (cover slip)
  • Oil immersion
  • Small beaker
  • Staining rack
  • Lighter
  • Parafilms/paper towel/absorbent paper
  • Dyes
  • Tissue paper
  • Alcohol 70% (sterilization)

Preparation and fixation of bacteria for staining

With a wire loop, a small drop of each of the broth cultures were placed on clean slides.

Aseptic technique was used for this step:

  • flame loop
  • remove cap and flame lip
  • remove sample
  • flame lip
  • recap tube
  • smear organisms on slide
  • flame loop

If a culture is taken from solid media, a small drop of liquid NaCl was place on the slide and thoroughly mix with it a small bit of the culture. The drop was spread on the slide to form a thin film. The slides were allowed to dry in the air or by holding them high above a Bunsen flame.



  1. Prepare smear on a clean
  2. Stain with crystal violet for 30 seconds.
  3. Rinse with
  4. Flood the f1lm with Grams iodine and allow it to act for 30 sec. Rinse with water.
  5. Decolorise with 95% alcohol.
  6. Rinse with
  7. Counter stain with safranin for 20-30 9. Rinse with water and blot dry.
  8. Examine under oil immersion objective.



In microbiology, differential staining techniques are used more often than simple stains as a means of gathering information about bacteria. Differential staining methods, which typically require more than one stain and several steps, are referred to as such because they permit the differentiation of cell types or cell structures. The most important of these is the Gram stain. Other differential staining methods include the endospore stain (to identify endospore-forming bacteria), the acid-fast stain (to discriminate Mycobacterium species from other bacteria), a metachromatic stain to identify phosphate storage granules, and the capsule stain (to identify encapsulated bacteria). We will be performing the Gram stain and endospore staining procedures in lab, and view prepared slides that highlight some of the other cellular structures present in some bacteria.


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