Workshop 1: AN INTRODUCTION TO MICROBES & BACTERIA

1. Name the six groups of microorganisms. What are the structural and functional
differences you can use to differentiate them?
Bacteria, archaea, fungi, protozoa, algae and viruses.

2. How do viruses differ from microorganisms?

Viruses are. Acellular, obligate intracellular parasites with genetic material and a
protein coat. Lack cellular structure and cant carry out metabolic processes
independently.

3. Discuss the distinguishing features between prokaryotic and eukaryotic cells.

Nucleus, membrane bound organelles, size, reproduction.

4. Match the structures in a prokaryotic cell to their functions:

B Cell wall A Attachment to surfaces

D Endospore B Maintains cell shape
A Fimbriae C Motility
C Flagella D Protection from adverse environment
E Capsule E Increases the virulence
I Pili F Extrachromosomal genetic elements
H Plasma membrane G Protein synthesis
G Ribosomes H Selectively permeable
F Plasmids I Transfer of genetic material

Case study

In the summer of 1998, approximately 8000 individuals gathered to celebrate a Catholic

priest's ordination in Minas Gerais, Brazil. Within hours of food consumption, 4000 patients
experienced acute gastroenteritis, and approximately 2000 overwhelmed Emergency
Departments of 26 local hospitals. 81 people were admitted to ICU and 16 of those patients
progressed on to develop irreversible multi-system shock and died while hospitalized. The
trace-back investigation implicated food preparers who were positive for enterotoxigenic
Staphylococcus aureus as the source of contamination.

1. Microscopic examination showed that Staphylococcus aureus displays dark violet

colour after gram stain. What is the gram staining? Is this a gram-positive or negative
bacteria? Why?

Gram staining is a technique used to differentiate bacterial species into two

groups.
 Positive gram staining = purple
Gram negative – pink

2. What is the shape and arrangement of Staphylococcus aureus?

Staphylococcus aureus is Gram-positive bacteria that are cocci-shaped and tend
to be arranged in clusters that are described as “grape-like.”

3. Describe other shapes and arrangement of bacteria.

Cocci – round
Bacilli – Rods
Spirilla – spiral
Vibrios – comma shaped
Spirochetes – helical

Arrangements
- Diplo – airs
- Strepto - chains
- Staphylo – clusters
- Tetrads – four
- Sarcinae – Cubed eight
-

4. Compare and contrast the structure and function of cell walls of Gram-Positive and
Gram-Negative bacteria.

Gram positive has thick peptidoglycan layer, teichoic acids. Function – protection,
structural support, Target antibiotics
Gram negative has thin peptidoglycan layer, Outer membrane, periplasmic space.
Function – protection, endotoxin, selective permeability.

5. Why is it important to know if a bacteria is Gram positive or negative clinically?

Antibiotic selection, infection site prediction.

6. Can Staphylococcus aureus form endospore? Explain whether endospore formation in

bacteria is a mode of cell reproduction. Of what advantage is an endospore to a bacterial
cell?

Endosporre formation is a survival strategy. Endospore formation does not involve

cell division. Instead it produces bacteria to endure extreme temperatures.

The following questions require you to watch the video on food poisoning (link can be
found under class activities).
7. Name other bacteria that may cause food poisoning.
 Workshop 2: MICROBIAL GROWTH

Answer the following questions:

1. At what pH do most bacteria grow best?

-5

2. On the basis of growth range of temperature, what are the 3 main groups in which
bacteria are classified?
- psychrophiles -20° - 10
- Mesophiles - 20 - 45
- Thermophiles – 45-80°

3. Describe the five groups of bacteria based on their oxygen requirements.

- Obligate aerobes – depend on oxygen to generate energy.
- Facultative Anaerobes – can grow in presence or absence of oxygen
- Aerotolerant Anaerobes – Tolerate presence of oxygen
- Microaerophiles – require low of oxygen for growth.

4. Why can high concentrations of salt or sugar be used to preserve food?

- due to their ability to create a hypertonic environment which inhibits the growth
of microorganisms including bacteria, yeasts, and moulds.

5. Explain how chemically defined media is different from complex media.

- chemical and complex media are both used to cultivate microorganisms.

6. What is the reducing media? What type of bacterial can be cultured using this media?
Reducing media – Contains reducing agents to create anaerobic conditions.
Purpose – cultures anaerobic bacteria that cannot survive in oxygen.

7.) For what purpose do differential & selective media are used? Name the commonly used
differential media.
- differential media is used to distinguish between microorganisms.

- Selective media inhibits the growth of certain microorganisms while allowing

others to grow.
-
 8. What is the method commonly used to obtain pure culture?
- Streak plate method. A small amount of mixed microbial culture is streaked onto
an agar plate in a specific pattern.

Case study (work in a group of 5-6 students)

Emma is a research assistant in a pharmaceutical company and she has recently joined a
research team investigating the efficacy of a novel antibiotics. Prior to animal studies and
consequential clinical trials they plan to test the effects of antibiotics on S. aureu in the
laboratory. Emma has been assigned to culture S. aureus cells for the experiment.

1. Emma starts to prepare the culture media. She is aware of the macronutrient requirement
and they are often listed as CHONPS (these are the chemical requirements required for
cell growth). What does each of these letters stand for? Explain why they are needed by
bacteria.

- Carbon – fundamental building block of organic molecules. Used to synthesize proteins, nucleic
acids, lipids and carbohydrates.
- Hydrogen – Essential for structure, especially in proteins and nucleic acids.
- Oxygen – Aerobic respiration, process to generate energy. Serves as terminal electron acceptor in
the electron transport chain.
- Nitrogen – Key component of proteins and nucleic acid (DNA and RNA). Required for synthesis of
enzymes, structural proteins and genetic material.
- Phosphorus – component in ^ and ATP and phospholipids (important for cell membranes.
- Sulfur – Found in amino acids and vitamins. Structure of protein.

2. She decides to start with six (6) S. aureus cells in a petri dish. If S. aureus has a
generation time of 60 minutes, how many cells would be found in the petri dish after five
hours?
192 cells. 6x2^5.

3. Explain how those bacterial cells multiple. In your understanding, how efficient is this
mode of reproduction compared with human reproduction?
- Replication of DNA
- Cell Elongation
- Septum formation
- Formation of daughter cells
- Cell separation

4. Draw a typical bacterial growth curve in the laboratory. Label and describe each of the
four phases.

5. In order to achieve the best outcome, the antibiotics should be applied at what stage of
the growth phase? Why?
- The optimal stage to apply bacterial cultures is the log phase as the bacterial
growth is exponential., as cells are dividing
-
 Workshop 3: CONTROL OF MICROBIAL GROWTH

1. The thermal death time for a suspension of B. subtilis endospores is 30 minutes in dry
heat and less than 10 minutes in an autoclave. Which type of heat is more effective and
why?

- Autoclave as the moisture in the autoclave creates a harsh environment for the
spores, causing them to undergo irreversible changes in their structure, therefore
leading to their distruction within a shorter period compared to dry heat.

2. Explain how Thermal death point (TDP) differs from Thermal death time (TDT).

- Thermal death point is the lowest temp that kills all microbes in liquid suspension
in 10 minutes.
- Thermal death point is the minimum time to kill all microbes in a liquid culture at
a given temperature.

3. Fill in the following table.

Method of Temp. Time Type of Preferred Type of Action

Control Heat Use against Microbe

Autoclaving 121 c 15min Moist Glass, metals.

heat
Hot Air Oven

Pasteurisation 63 or 30m Moist Milk

72° heat

4. What are five (5) factors, which influence effective control of microbes and briefly
explain them?
Number of microbes (microbes increase, longer time),time of exposure (long
exposure leads to a decrease of microbes), microbial characteristics, environmental
influences,
Environmental influences, temperature.

5. Discuss the differences between gamma and ultraviolet radiation. Give one example of
their application. What is their anti-microbial effect due to?

Gamma rays can penetrate materials and are used for heat sensitive
pharmaceutical products e.g., prepacked dressing packs, needles syringes, catheter.
Ultraviolet rays are non-ionising ( part of sunlight, must have direct exposure as
they cannot penetrate, damaging to the human eye and causes skin cancer.
6. How can heat-sensitive solutions, such as glucose-minimal salts (microbial growth)
medium be sterilised?
Use filtration and radiation.

7. Why is food treated by pasteurisation when it does not achieve sterilisation?

As pasteurisation kills pathogenic organisms or those that cause spoilage without
altering flavour or destroying important molecules.
Case study

Sally is a manager of the infection control unit in a public hospital. During her inspection this
morning, she found that surgical instruments had been autoclaved at 108 C. She immediately
called off all scheduled surgeries on the day due to the concern of potentially exposing
patients to risks of infection.

1) Has Sally made a right decision? Justify your answer?

Yes as the sterilization temperature through autoclaving should be 121°.

2) Explain how sterilisation is different from disinfection and sanitization.

Sterilisation is the removal or destruction of all microorganisms in areas or on
objects, whereas disinfection is the removal or destruction of a large number of
microorganisms in areas or on objects (however it does not inactivate endospores
(sterilising does). Sanitization only reduces microbial numbers to a safe level.

3) What are the commonly used methods of sterilisation? Which one is the preferred method to
cleaning surgical instruments?
- Autoclaving, as they are highly effective in killing a wide range of microorganisms
including bacteria, viruses and spores.

4) Can glutaraldehyde be used to sterilize surgical instrument?

Yes, glutaraldehyde (it’s a aldehyde), it kills bacteria including macrobacteria, and viruses in 10
mins, endospores after 3-10 hours.

5) Name other two main chemical sterilizers?

Formaldehyde, chlorine, Glutaraldehyde.

6) Rank the microbes based on their resistance to antimicrobial treatment (high to low): Gram
+ve, Gram –ve, enveloped viruses, non-enveloped viruses, fungi, endospores.

1.) Endospores
2.) Non – enveloped viruses
3.) Gram negative bacteria
4.) Enveloped viruses
5.) Gram positive bacteria
6.) Fungi
Discussion should include
Contamination of ginger garlic.
Clearsomes were not measured in comparative results.
Talk about how it wasn’t resistant but it developed resistance, clearzome was visible
multiple generations developed resistance to colonise the area.