MICROBIAL BIOLOGY LABORATORY

REPORTS

MIBI LAB REPORT

REPORT 1: MEDIUM PREPARATION
1. Why do we have to incubated newly prepared medium at 37oC before storing in the fridge?  to
kill unwanted microbes for sterilization
2. Why unused sterile medium should be then stored in the fridge rather than RT condition?
 because most bacteria cannot grow well in cold temperature condition
3. Why 70% EtOH is used as disinfectant, not EtOH with
a. Higher concentration (90%,100%)  higher concentration is not effective in killing microbes, it will
evaporate fast.
b. Lower concentration (40%, 50%)  because it is usefulness for disinfection drop sharply
4.
a. What is full name of LB medium?  Luria – Bertani
b. List name and amount of the ingredients to make 1 L of LB agar medium.
+ Sodium Choloride: 10g + Tryptone (Casein protein): 10g
+ Yeast extract: 5g + Agar: 15g
+ Distilled water up to 10 mL
5. Show calculation for getting the % of concentration of agar:
 % agar = 15/1000 = 1.5%
6. Show calculation for obtaining the amount of agar needed to make 800 mL LB agar medium.
15g agar  1000mL LB agar
?  800mL LB agar
 ? = (800 x 15)/1000 = 12g agar
7. Why reused sterile petri dish might have higher risk of getting contamination than using
disposable sterile dishes?  because some bacteria can adhere on the surface of the glass and effect the
next results
8. Name + processing of following nutrients
- Casein hydrolysate: polypeptides  process of casein separation, hydrolysis, concentration, pray drying.
- Tryptone: assortment of peptide  form by the digestion of casein by the protease trypsin.
- Peptone: excellent source of acid amine  obtained by enzymatic digestion or acid hydrolysis of natural
product.
9. Show calculation how to make 600 mL of 70% EtOH from 95% EtOH and water?
C1 x V1 = C2 x V2
From 600mL of 70% EtOH
0.95 x V1 = 0.7 x 600 => V1 = 442 mL
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V(H2O) = 600 – 442 = 158 mL
So take 442 mL of EtOH fill with water until it reach 600Ml

REPORT 2: ASEPTIC TECHNIQUE/ CULTURE TECHNIQUE SUBCULTURE

1. What is the purpose of flaming in the aseptic technique?
- Prevent contaminant to sample (before); prevent transferring microorganism to the environment (after)
2. Provide definitions for the following term.
- Bacterial colony: each cell on the agar gel grow, replicate and form a visible clump. They all have same
genetic.
- Inoculumn: indicate the origin amount of cell we introduced.
- Bacterial culture: is medium that have microorganism (bacteria)
- Culturing medium: is the nutrient preparations that are used for culturing microorganism. There are 3
physical forms: liquid media, semisolid media and solid media.
3. In all routime lab work, agar will have lables on the bottom. Why?
- To avoid mistakes between plates when open the plates.
- Easy to work at the bottom, we can see our work at the top.
4. Why can a single colony on the agar plate be used to start a pure culture?
- Because a single colony contain many cells that have the same genetic mateial. They are in the same
species  pure culture.
5. In which situation that a distinct colony seen on the agar durface does not represent for the
growth of 1 cell?
- The areas are not inplanted any bacteria to the agar surface.
- The colony contain dead cells, which cannot grow on the medium.
6. What are the 2 purposes of sub-culturing?
- To isolate a pure colony from different species for obtaining pure culture.
- To maintain the survival of bacteria.
7. How would you determine whether the culturing medium supplied by your instructor are sterile?
- Agar plate: it is pure without strange things (e.g. white dot) on the agar surface. We check plate, is it
damage or not and check the smell of the plate (agar gel)
- Broth: the white liquid in tube is purified with no seeing, strange things inside which means the medium
is not cloudy.
8. How would you regconize/check the presence of contaminant in your bacterial culture?
a) You grow your bacterium on the agar plate?
- If an agar plate has bacteria grown on the surface, it will produce abnormal odour on the surface.
Colonies have different colors and shapes.
b) You grow your bacterium in broth?
- If a culture has grown in broth, the medium’s turbidity is increased, meaning that it should be hazy and
cloudy. Check for other colors, too.
9. Why contamination can be minimized if you perform bacterial inoculation near the flame?
- Because the flow of the alcohol flame will make the air around hot, which will avoid the contaminant
outside go into working area.
10. Dicussion for Lab 5 results:

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a) Give a comment about the class Standard Plate Count result:
- DF = 10-7. After a week, we count 29 big colonies and 1 small colonies.
- There are 2 brown small colonies of other species  our plate is contaminated.
- For our class: DF = 10-6 is the only plate is not contaminated, but the size of the colony still bigger than
normal. To explain, there are many cells stick there or many colonies clump together  form big size.
This spread technique may not be done properly.
b) Which plate (e.g. which Dilution Factor) should you use to estimate the number of living E.coli
cells in the original culture? Explain your choice.
- The plate with DF = 10-6 should use to estimate the number of living E.coli cells because it is not
contaminated.
c) Use the plate (Q10.b) to show calculation for:
 Estimate the number of living cells per mL of the original culture:
DF = 10-6
#CFU = 66
Vplanting = 100 uL = 0.1 mL
¿CFU 66
The number of living cell per mL: #cell = × V original culture= −6 ×1=6.6× 107 mL
DF × V planting 10 × 0.1
 Estimate the number of living cells per uL of the original culture:
1 mL = 1000 uL

6.6 ×107
The number of living cell per uL: #cell/uL ¿ =66000 mL
1000
d) Give comment about the culturing results from your soil sample:
- Group 1, 3, 5: heat – treatment
- Group 2, 4: no het – treatment
- The plate has different colors, has different microbes. It contains bacteria (yellow) and fungi (dark
brown). The plate has a lot of species. We don’t have heat – treatment, which can kill microbes that live
more than 700C.

REPORT 3: USE OF THE MICROSCOPE WET MOUNT/ SMEAR PREPARATION GRAM

STAINING
1. Why is the low-power objective placed above the stage when the microscope is stored or carried?
- Because the low-power objective is further away from stage than the others, so it is less likely to get
scrapped during handling.
2. Why is oil necessary when using x100 objective lens?
- As magnification increases, resolving power decreases. Oil is used to increase the resolving power of
microscopes by immersing both the objective lens and the specimen in a transparent oil of high
refractive index (chiết xuất).
3. What is the function of substage condenser?

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- Gathers light from the microsope light source and concentrate it into a cone of light that illuminates the
specimen with uniform – intensity over the entire viewfield.
4. What is function of Iris diaphragm?
- Regulate the amount of light entering the lens system.
5. Why are unstained bacteria more difficult to observe than stained bacteria?
- Bacteria is so small. Unstained bacteria has refractive index close to water, so there is not much contrast.
Staining add contrast and make them easier to see. Bacteria can move around fast, making it hard to see
them under the microscope.
6. List 2 purposes of heat – fixation step.
- Kill bacteria without destroying it. Living cells can contain enzymes that can break down the structure
of the bacteria.
- Fix the bacteria to the side.
7. How does true motility different from Brownian movement?
- Brownian movement is a small vibrational movement due to the bombardment of water molecules.
- True motility is self-directed and more than simple vibrations back and forth.
8. How would you define a properly prepared bacterial smear?
- A properly bacterial smear would ean the bacteria are evenly spread out and properly fixed. This way
they don’t wash off with DI water.
9. Why would you use an inoculating needle when making smears from solid media?
- To retrieve the specimen so not too much is transferred.
10. Name the reagents and state the purposes of using of each of the following:
a) Mordant: Iodine solution: increase the interaction between the bacterial cell and the dye, so that the dye
is more tightly bound or the cell is more strongly stained.
b) Primary stain: crystal violet stains the microbes that are both Gram(+) and Gram(-).
c) Decolorizer: 95% ethanol: remove the dye out of the cell wall.
d) Counterstain: Safranin: stain the colorless. Gram(-) bacteria is pink but does not alter the dark blue color
of the Gram (+) bacteria.
11. For Gram staining procedure, timing the application of the colorizer to your bacterial smear is
important in getting correct result. Explain.
- If the time colorizing is too short, alcohol cannot remove all the crystal violet in Gram(-), causes that
Gram(-) after staining will have purple color like Gram(+).
- If the time for colorizing is too long, alcohol can remove the crystal violet in Gram(+) cell wall, causes
that Gram(+) will have pink color after staining, which like Gram(-)
12. What part of bacteria cell is mostly involved in Gram stain?
- Peptydoglycan cell wall.
13. Explain why Gram(-) can lose the stained color of crystal violet – iodine complex upon
decolorization?
- In Gram(-), the peptydoglycan is thicker than in Gram(+). Alcohol can dissolve the crystal violet stains
and remove it out of the cell wall.
14. Why shoud you use young culture for Gram staining?
- Young culture must be used so the cystal violet can stick to the cell walls of Gram(+) bacteria. The cell
walls breakdown in cold culture and staining process is not accurate.
15. What is meant by Gram variable?
- Gram variable means the bacteria may stain either Gram(-) or Gram(+).
16. Draw Gram stain results fro 2 bacteria you observed under imcroscope. Include your lable.
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Group 4 Group 2

Gram(+) that have purple color collected from soil, Staphylococcus aureus.
white bacteria. It has spherical shape (staphy lococi)
The bacteria has rod shape. It has purple color  Gram(+)
 Brownian movement:
- Is the random movement of particles suspended in a fuild.
- Is vibrational movement caused by invisible molecules bombarding bacterial cells. If the only movement
you see is vibrational and not directional, the organism is non-motile.

REPORT 4: EFFECTS OF FACTORS PREPARATION & CARE OF STOCK CULTURE

1. Why are strict anaerobes unable to tolerate O2 while other groups of microorganisms can?
- Because they lack of catalase and superoxide dismutase and thus are killed by oxidative from oxygen
redicals.
2. Define “reactive oxygen species (ROS)”:
- ROS are chemically reactive chemical species containing oxygen (both reactive molecules and free
radicals)
3. Redical ROS: superoxide anion

4. Non-radical ROS: hydrogen peroxide H2O2

5. What are effects of ROS to cellular activities and function if they are present in cell at excessive
level?
- Effect: cause “oxidation – stress” to cell
- Function: damage nucleidacid causing perooxidation of lipid, oxidation of protein enzyme inhibition,
activation of programmed cell death (PCD)
6. Name 3 strategies that living organisms can use to scavenge ROS out of cell.
- Use superoxide dismutase (SOD) catalyse H2O2 to water and O2 complete the detoxification initated.
- Selenium/catalyze H2O2 as well as organic peroxides to alcohol.
7. Name 1 metabolic pathway/ biological process taking place in living cells that produce ROS as
intermediate product? Also provide name(s) of ROS.
- In oxidative phosphorylation, mitochondria produce superoxide in the electron transport chain.
- In photosynthesis of plants, single oxygen (O2) is produced.
8. Why do we need to prepare reserve stock culture?
- Use it as a back up in case of contaimination of working stock.
9. Name chemical(s) are generally added in reverse stock culture to maintain better vability of
microorganisms when they are kept at freezing temperature (-800C) condition.
- Glycerol
10. What culture stock do we use to identify one unknown bacteria?
- We use the workig stock.
11. Give scientific description about your studied microorganism in relation to the parameter
(oxygen/temperature) you are studying.
 Baccilus:
- Rod-shape, endospore-forming

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- Aerobic or facult actively anaerobic and are mesophile (optimal of 25 – 350C)
 E.coli:
- Rod-shape, falcutative anaerobic and they are Gram (-)
- Mesophile (optimal 20 – 370C)

REPORT 5: BACTERIA POPULATION COUNTS

1. Why is it necessary to perform a plate count in conjunction with the turbidity measurement?
- Turbidity give quick count of biomass (dead and alive cells), while SPC can give you the number of
colonies that across from a single cell. Turbidity can evaluate the culture status: young or old. While
SPC cannot give the status of the culture.
- SPC give the information that culture is containminated or not, but the turbidity can’t. so we must
combine these 2 methods.
2. What is a Colony-forming Unit (CFU)?
- It describes how many bacteria are put into a petridish to grow into visible groups.
3. For Standard Plate Count method, if your culture has been diluted up to 1 000 000 times yet you
are still unable to count CFU (due to clumping), suggest 2 ways that you can overcome this
problem.
- Way 1: Keeping the suspension in an ice both, in plastic tubes and by using diluent without Calcium and
Magnesium. Always mix throughly before sampling.
- Way 2: gently pipetting the sample up and down a few times.
4. For bacterial enumeration, list at least 2 problems might arise from pipetting very small volume of
culture.
- Tips have to be pushed powerfully onto pipette cone in order to achieve efficient tip fit.
- Banana-shape tips make it difficult to fill a plate with multichannel pipettes.
- Pipetting of volumes below 1 uL on a solid surface is impossible because the liquid drop stick to the
outside of the tip.
5. You are supplied with 10 mL of E.coli culture (E.coli grown in LB broth) and 50 mL of sterile Lb
broth. Showing calculation to dilute 10 000 times by single-step dilution method.
- Prepare a tube that contain 9999 uL of sterile LB broth.
- Shake the culture of E.coli and transfer 1 uL to the tube of 9999 uL sterile LB broth. We have 1/10 000
dilution of the original one.
6. You are supplied with 10 mL of E.coli culture (E.coli grown in LB broth) and 50 mL of sterile LB
broth. Showing calculation to dilute 10 000 times y serial-step dilution method, each time dilute
10-fold.
- Prepare 4 eppendorf that contain 9 mL od sterile LB broth.
- Shake the culture of E.coli transfer 1 mL to the 1st tube of 9 mL of sterile LB broth by using a sterile 1.1
mL pipette. We have a 1/10 dilution.
- Shake the 1st tube well. Then, transfer 1 mL of the 1st tube to the 2nd tube, we have a 1/100 dilution.
- Shake well and transfer 1 mL of the 2nd tube to the 3rd tube to get 1/1000 dilution.
- Shake well and transfer 1 mL of the 3rd tube to the 4th tube to get 1/10 000 dilution.

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MICROBIAL BIOLOGY LABORATORY
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