Introduction:
Top 10 Techniques Every Microbiologist Should Know, the fascinating science of tiny organisms, plays a pivotal role in understanding our world and improving our lives. Whether you’re a student stepping into the vast microbial universe or a seasoned microbiologist looking to refresh your knowledge, mastering fundamental techniques is crucial. Here, we’ve compiled the top 10 techniques every microbiologist should know, designed to be engaging, informative, and accessible to all.
Here, Top 10 Techniques Every Microbiologist Should Know:
1. Aseptic Techniques:
These techniques are paramount for creating a sterile environment to prevent contamination from unwanted microbes. The main importance of aseptic techniques in microbiology is creating suitable controlled conditions to prevent or at least reduce the contamination from other microbes (that entering unsterile sources like water or air or dust) as much as possible so we can grow and study specific microorganisms.
2. Staining Techniques:
Microbes are often transparent, making them invisible to the naked eye. Staining techniques utilize dyes to color the cells, allowing for visualization under a microscope. Different staining methods reveal specific cellular structures or differentiate between various microbial types.
Staining techniques in microbiology depend on dying or coloring the cells and other structures to be highlighted for viewing often with the aid of microscopes.
Top types of Staining Techniques in Microbiology
- Simple Staining
Simple staining depends on using only single dye like methylene blue and crystal violet which are used to determine the shape, size and arrangement of the cells.
The steps are:
1- Apply a single dye to a fixed smear in order to color the microorganisms.
2- Cover the fixed smear with stain and wait for a specific period.
3- Then, wash off the solution with water and dry the slide.
- Differential Staining
This type of staining technique is used to differentiate microorganisms based on the properties of staining. More than one dye is used to distinguish organisms. For example: gram staining, acid-fast staining, endospore staining, and metachromatic staining.
- Gram Staining
It is one of the most important and widely used differential staining techniques in microbiology. It is called gram staining after the Physician Christian Gram introduced it in 1884 and it is used to divide bacteria into two categories (Gram positive and Gram negative).
The following figure illustrates Gram staining procedure
1- Apply the basic dye (ex: crystal violet) to the smear to stain it.
2- Add iodine solution (as it increases the interaction between dye and cells so it stains sharply).
3- Wash the smear with ethanol or acetone to be decolorized.
4- The previous step makes the differential aspect of Gram stains. Gram positive bacteria retain crystal violet and become colourless.
Finally the smear is counter-stained with a basic dye that must be different in color from Crystal violet like safranin which changes the colour of Gram negative bacteria from pink to red and leaves the colour of Gram positive bacteria dark purple.
3. Culturing Techniques:
Culturing is the process of growing microorganisms in a controlled environment using a culture medium that provides essential nutrients for their survival and multiplication. This technique allows for isolation, identification, and further analysis of specific microbes. Culturing Techniques are among the top 10 techniques every microbiologist should know.
4. Isolation Techniques:
In nature, microbes exist in mixed communities. Isolation techniques aim to separate a single type of microbe from a mixed culture. This enables the pure culture to be studied and characterized without interference from other microbial species.
We can isolate different types of pathogens from body tissues or fluids such as blood, urine, pus, sputum, faces, spinal fluid, stomach fluids, bile and pleural fluids. Isolation Techniques are among the top 10 techniques every microbiologist should know.
The following isolation methods are the most common and employed to isolate microbes from mixed cultures:
- Streaking
- Plating
- Dilution
- Enriched procedure, and
- Single cell technique.
5. Differentiation Techniques:
Not all microbes are created equal. Differentiation techniques help distinguish between different types of microorganisms based on their morphology, staining properties, biochemical characteristics, or growth patterns. Differentiation technique is among the top 10 techniques every microbiologist should know.
Beyond the Top 5
The world of microbiology encompasses a vast array of techniques. Here are some additional methods that play a crucial role in specific areas of study:
6. Microscopy:
Microscopy is the cornerstone of microbiology, allowing scientists to observe the size, shape, and morphology of microbes in minute detail. Light microscopes are widely used, while electron microscopes provide even higher resolution for in-depth cellular analysis.
7. Biochemical Tests:
These tests assess the metabolic capabilities of microorganisms. By analyzing how microbes utilize different substrates or produce specific enzymes, scientists can gain valuable insights into their identification and classification. Biochemical test is among the top 10 techniques every microbiologist should know.
Fast and simple biochemical tests are used to identify infectious pathogens. For example, in the case of bacterial identification, the tests depend on the presence of acids, gasses and alcohols to detect the growth of bacteria.
Examples of Biochemical tests used for bacterial identification:
- Catalase Test
- Antibiotic Sensitivity Test
- Coagulase Test
- Indole Test
- Oxidase Test
- CAMP Test
- Starch hydrolysis test
- Bile Esculin Agar Test
- Taxos P (optochin sensitivity testing)
- Taxos A (bacitracin sensitivity testing)
- Nitrate Broth
- Methyl Red / Voges-Proskauer (MR/VP)
- Urease test
- Sulfur Indole Motility Media (SIM)
8. Polymerase Chain Reaction (PCR):
PCR is a powerful molecular technique used to amplify specific DNA sequences. This allows for the detection and identification of microbes even when present in very low numbers.
Polymerase chain reaction (PCR) procedure is the most used molecular technique to determine and study microbes. If we compare PCR technique to other techniques like sequencing , we will find that PCR is fast, accurate, definitive and reliable.
9. Antimicrobial Susceptibility Testing:
This technique determines the effectiveness of various antibiotics against a particular microorganism. This information is critical for guiding treatment decisions in cases of infectious diseases.
10. Immunological Techniques:
These techniques exploit the immune system’s response to identify and differentiate between microbes. Techniques like serological assays can detect antibodies produced against specific pathogens, aiding in diagnosis and epidemiological studies.
By mastering these techniques, microbiologists unlock a world of possibilities. These tools empower them to investigate the intricate world of microbes, leading to advancements in various scientific disciplines and ultimately contributing to a better understanding of our world.
New Techniques in Microbiology (MALDI-TOF Mass Spectrometry)
Rapid identification of microorganisms in the clinical microbiology laboratory by using MALDI-TOF MS is considered one of the important new microbiology techniques and can be of important and great value for the process of optimal patient management strategies selection for infections caused by viruses, bacteria, mycobacteria, fungi, and parasites.
In the first experiment of MALDI-TOF MS, it succeeded in identifying bacteria directly from the whole colonies dependent on protein biomarkers. After that a big number of developments and advancements have been made on whole-organism MALDI-TOF MS. The protein biomarkers which are measured in microorganisms mass spectrometry are considered as highly expressed proteins and they are responsible for functions like ribosomal translation and transcription.
These diagnostic methods in microbiology that are used for the rapid identification of microorganisms in clinical samples enables expedient de-escalation from broad-spectrum agents to targeted antimicrobial therapy. The torn to this specific therapy decreases risks of antibiotics, normal flora disruption, selective pressure and toxic side effects. There is a necessary need for new technologies and advancements in microbiology, specially in clinical microbiology and particularly for infections of the bloodstream that are related to the highest of all infections.
Advancements in Microbiology
Now we will review some of the latest discoveries and recent developments in microbiology:
Dormant strains of bacteria that have previously adapted to cope with certain temperatures are switched back on during climatic change
“Understanding the relative importance of acclimation, adaptation and species sorting in the assembly and turnover of microbial communities is key to determining how quickly they can respond to temperature changes. Until now, a mechanistic basis of these community-level responses had not been discerned ,” concludes senior author Thomas Bell, Professor of Microbial Ecology at the Georgina Mace Centre for the Living Planet, Imperial College London. “We have found that the resuscitation of functional diversity within a microbial community can allow the whole community to survive in response to temperature changes. Further studies on other microbial communities — such as those residing in water — will support more accurate predictions of the effects of climate change on different ecosystems.”
