Microscopy analysis applied to activated sludge matrices is an essential tool for understanding the characteristics of the effluent, identifying the root cause of operational issues, and assessing the current conditions of the treatment system.

In this article, you will learn the three main steps for conducting a high-quality microscopic analysis. With this information, you will be able to draw robust conclusions about the system's current situation.

Step 1 – Analysis of Floc Formation

Place the prepared sample slide on the microscope stage and use 10x or 20x objectives to analyze floc quality. The first step is to assess the characteristics of the flocs:

• Shape: Rounded or Irregular
• Strength: Firm or Weak
• Structure: Compact or Open

To evaluate the structural strength, perform the pen test by gently applying pressure with the tip of a pen on the coverslip. Flocs classified as firm maintain their initial structure without disintegrating.

At this stage, try to identify any potential issues in floc formation, which may include:

• Dispersed Growth: Excess free bacteria in the solution forming small cell clusters.
• Filamentous Bulking: Excess filamentous bacteria extending outside the floc and/or opening its structure.
• Pin Floc: Small-sized flocs that have undergone mechanical shear.

Microphotograph of an ideal floc: rounded shape, firm strength, and compact structure. Phase contrast microscopy, 100x magnification.

Step 2 – Microfauna Analysis

This stage involves the identification and quantification of microfauna, which includes various groups of microorganisms such as:

• Protozoa: Flagellates, amoebas, testate amoebas, floc-predating ciliates, sessile ciliates, carnivorous ciliates, and free-swimming ciliates.
• Micrometazoa: Rotifers, tardigrades, annelids, and nematodes.

Use a 40x objective for better observation and accurate identification of microbial groups. During this analysis, note which groups predominate in the sample and count each identified group by scanning the entire coverslip area.

The greater the diversity and active population of microorganisms, the healthier the microbiota will be.

Microphotograph of free-swimming ciliates. Phase contrast microscopy, 400x magnification.

Additionally, the appearance of specific organism groups based on the type of available nutrients can help estimate the sludge age through the relative predominance diagram.

For example, flagellates feed only on dissolved nutrients—large amounts of flagellates may indicate young sludge age and/or a highly disturbed system.

Relative Predominance Diagram.

Source: Class, 2007.

Step 3 – Filamentous Bacteria Analysis

The final step is the evaluation of filamentous bacteria. The identification and quantification of these filaments directly influence sludge settling capacity and foam formation. When present in adequate amounts, filamentous bacteria contribute positively to COD reduction and floc structure formation by adding size and rigidity.

On the other hand, excessive proliferation of these bacteria compromises floc density, resulting in lower sedimentation capacity. In such cases, sludge becomes bulky, light, and swollen, which impairs separation in the secondary clarifier and can lead to solids carryover.

To identify the genus of filamentous bacteria, it is recommended to use complementary techniques such as Gram and Neisser staining, along with morphological analysis—including cell diameter and shape. Together, these allow more accurate identification with the help of a specific dichotomous key.

Observation should be done using an oil immersion objective at 100x magnification. Based on the observed morphology, classify the quantity of filaments per floc using the scale below:

GRADE 1 – Rare: 1 to 4 filaments per floc

GRADE 2 – Common: 5 to 20 filaments per floc

GRADE 3 – Abundant: 21 to 50 filaments per floc

GRADE 4 – Excessive: More than 50 filaments per floc

Dichotomous key.

Microphotograph of filamentous bacteria with morphology similar to the genus Nocardia. Phase contrast microscopy, 1000x magnification.

Observing, interpreting, and acting based on what we see under the microscope turns visual data into strategic decisions for operations.
If you want to learn more about how to effectively implement this practice in your plant or operation, contact Genetica. We’ll be happy to help your team see the process with new eyes!