The objective of this experiment is to extract C. beijerenckii from soil samples, then calculating the amount of biobutanol production

This experiment will follow these 7 steps:

1. Preparation and storage of media (RCM and P2)
2. Sample collection and Pasteurization
3. Growth of bacteria (random unknown yet strains of Clostridium)
4. Inoculation of strains on plates 
5. Morphological, Biochemical and Physiological Characterization of the Isolates
6. Fermentation of C.Beijerinckii 
7. Testing of butanol production

1- Sample Collection:

 C. beijerinckii is mainly found in agricultural soil (Mohamed Hemida Abd-Alla et al). Therefore, we are going to collect our samples from agricultural areas. C. beijerenckii can be isolated from samples collected from the rhizospheres of potato, onions, cucumber, strawberry and parsley. 

The collected samples will meet the following criteria:

1- 10 cm depth

2- 10 g sample mass

After collecting the samples, we must use them immediately, otherwise they must be stored in -20 degrees Celsius

2- All required materials:

Media:

    1.RCM Medium:

• Yeast extract
• Peptone from casein (Tryptone)
• Meat extract
• Glucose
• Starch
• Sodium chloride (NaCl)
• Sodium acetate
• L-Cysteine hydrochloride
• Agar

   2.P2 Medium:

• Yeast extract
• KH₂PO₄ (monopotassium phosphate)
• K₂HPO₄ (dipotassium phosphate)
• Ammonium acetate
• MgSO₄·7H₂O (magnesium sulfate)
• MnSO₄·H₂O (manganese sulfate)
• FeSO₄·7H₂O (iron(II) sulfate)
• NaCl (sodium chloride)
• Para-aminobenzoic acid (PABA)
• Thiamin (Vitamin B1)
• Biotin (Vitamin B7)
• Glucose (or glucose–xylose mix, glucose–acetone, or corn stalk hydrolysate as carbon source)

Chemical Components / Reagents

Staining reagents

• Crystal violet
• Gram's iodine
• Ethanol or acetone-alcohol (decolorizer)
• Safranin
• Malachite green
• Immersion oil

Biochemical test reagents

• 3% hydrogen peroxide (H₂O₂)
• Kovac's reagent
• Rifampicin (serial dilutions: 0.125–16 µg/mL)
• Lucas reagent (ZnCl₂ in concentrated HCl)

Growth media / broths

• Tryptone broth (tryptone 10 g/L, NaCl 5 g/L)
• SIM medium (Sulfur–Indole–Motility)
• Christensen's urea agar
• Urea broth
• Nutrient gelatin (deep tubes)
• Litmus milk
• Skim milk powder (10% w/v)
• P2 medium (for butanol fermentation)

Carbon sources (for fermentation/hydrolysis tests)

• Glucose (1% standard; 60 g/L for butanol trials)
• Xylose
• Sucrose
• Lactose
• Starch

General reagents / solutions

• Sterile distilled water
• Sterile saline
• Phenol red (pH indicator)
• Bromocresol purple (pH indicator)
• NaCl
• Phosphate buffer

Devices & Equipment

Microscopy

• Glass slides
• Microscope with 100× oil immersion objective

Inoculation & loop work

• Inoculating loop (metal, for flaming)
• Sterile straight inoculating needle
• Sterile wooden sticks or plastic loops

Heating

• Bunsen burner / flame
• Staining rack above boiling water or hot plate

Incubation & anaerobiosis

• Incubator (35–37°C)
• Anaerobic incubation system/chamber

Measurement & analysis

• McFarland standard (for suspension standardization)
• Durham tubes (for gas detection)
• Spectrophotometer / OD600 reader
• pH meter
• GC (Gas Chromatography) or HPLC (for butanol confirmation)

General lab items

• Test tubes and tube caps
• Autoclave (for sterilization)
• Refrigerator or ice bath (for gelatin liquefaction cooling step)
• Absorbent paper (for blotting slides)
• Well plates

- Media preparation:

2 media were used 

-1st) RCM ( Paula et. al., "A two-phase Model for ABE Fermentation with a Modified Clostridium Acetobutylicum Strain", CEt, VOL. 110, 2024): 
Yeast extract 3.0 g/L, Peptone from casein (Tryptone) 10.0 g/L, Meat extract 10.0 g/L, Glucose 5.0 g/L, Starch 10.0 g/L, Sodium chloride 5.0 g/L, Sodium acetate 3.0 g/L, L-Cysteine hydrochloride 0.5 g/L, and Agar 12.5 g/L.

-2nd) P2 medium:(Butanol–isopropanol fermentation with oxygen‐tolerant Clostridium Beijerinckii XH29)

In a 100 mL flask, 30 mL
medium supplemented with one carbon source (either glucose, glucose–xylose mix, glucose–acetone, or corn stalk hydrolysate)

 1 g/L yeast extract was sterilized at 115 °C for 15 min.

Then, upon cooling to room temperature, 0.3 mL of each filter-sterilized P2 stock (solution was added: (1) Buffer: 50 g/L KH2PO4; 50 g/L K2HPO4; 220 g/ L ammonium acetate; Mineral: 20 g/L MgSO4•7H2O; 1 g/L MnSO4•H2O; 1 g/L FeSO4•7H2O; 1 g/L NaCl; and (3) Vitamin: 0.1 g/L para-aminobenzoic acid; 0.1 g/ L thiamin; 0.001 g/L biotin)

The first medium was used for culturing strains, the RCM  

the second medium was used for fermentation.

- Methods: (detailed procedures)

A- Gram Staining:

Procedure(steps)

1. Slide preparation

1.     Take a clean, dry glass slide.

2.     Label the slide with the sample name.

3.     Place one small drop of sterile distilled water or saline on the slide.

4.     Flame the inoculating loop until red hot.

5.     Allow the loop to cool.

6.     Take a very small amount of bacterial colony.

7.     Mix the bacteria with the drop of water on the slide.

8.     Spread gently to make a thin smear

9.     Leave the slide to air dry completely.

Important:
The smear should be thin. A thick smear can give false results

2. Heat fixation

1.     After the smear is completely dry, pass the slide quickly through the flame 2–3 times.

2.     Do not overheat the slide.

Purpose of heat fixation:

• kills bacteria;
• fixes the bacteria to the slide;
• helps the stain enter the cells. 

3. Crystal violet staining

1.     Cover the smear completely with crystal violet.

2.     Leave for 1 minute.

3.     Rinse gently with distilled water.

At this step, all bacteria become purple.

4. Gram’s iodine

1.     Cover the smear with Gram’s iodine.

2.     Leave for 1 minute.

3.     Rinse gently with distilled water.

Iodine acts as a mordant.
It forms a crystal violet–iodine complex inside the bacterial cells.

5. Decolorization

1.     Add ethanol or acetone-alcohol to the smear.

2.     Decolorize for about 10–20 seconds, or until the runoff becomes almost clear.

3.     Immediately rinse with water to stop decolorization.

This is the most important step.

If you decolorize too much:

Gram-positive bacteria may falsely appear pink.

If you decolorize too little:

Gram-negative bacteria may falsely appear purple.

6. Safranin counterstain

1.     Cover the smear with safranin.

2.     Leave for 30–60 seconds.

3.     Rinse gently with distilled water.

Safranin stains Gram-negative bacteria pink/red.

7. Drying

1.     Blot the slide gently with absorbent paper.

2.     Do not rub the smear.

3.     Let the slide dry completely. 

8. Microscopic observation

1.     Place one drop of immersion oil on the stained smear.

2.     Observe under the microscope using the 100× oil immersion objective.

3.     Record:

• color;
• shape;
• arrangement;
• presence or absence of spores.

Interpretation

B-ENDOSPORE STAINING:

1. Prepare the smear

1.     Take a clean glass slide.

2.     Place one small drop of sterile water or saline on the slide.

3.     Using a sterile loop, take a small amount of bacterial culture.

4.     Mix it with the drop and spread to make a thin smear.

5.     Let the smear air dry completely.

Important:
For endospore staining, it is better to use an older culture, around 48–72 h, because spores are produced when bacteria face stress or nutrient limitation.

2. Heat fixation

1.     After the smear is dry, pass the slide quickly through the flame 2–3 times.

2.     Do not overheat. 

3. Stain with malachite green

1.     Place the slide over a staining rack above boiling water or on a gentle hot plate.

2.     Cover the smear with malachite green.

3.     Heat gently for about 5 minutes.

The stain should steam, but it should not boil or dry completely.

If the stain begins to dry, add more malachite green.

Why heat is used:
Endospores have a resistant coat, so heat helps malachite green enter the spore.

4. Rinse with water

1.     Remove the slide from heat.

2.     Let it cool for a short time.

3.     Rinse gently with distilled water.

Water removes malachite green from vegetative cells, but spores remain green.

5. Counterstain with safranin

1.     Cover the smear with safranin.

2.     Leave for 30–60 seconds.

3.     Rinse gently with distilled water.

Safranin stains the vegetative cells red/pink.

6. Dry the slide

1.     Blot gently with absorbent paper.

2.     Do not rub the smear.

3.     Let it dry. 

7. Microscopic observation

1.     Add one drop of immersion oil.

2.     Observe using the 100× oil immersion objective.

3.     Record:

• cell shape;
• spore color;
• spore position;
• spore shape.

Interpretation

For Clostridium spp., you may observe:

Green oval spores inside or outside pink/red rod-shaped cells.

The spores may be:

C-CATALAYSE TEST:

1. Prepare the slide

1.     Take a clean, dry glass slide.

2.     Label the slide with the isolate code.

3.     Place the slide on a clean bench surface. 

2. Add hydrogen peroxide

1.     Add 1 drop of 3% H₂O₂ onto the slide.

2.     Use fresh hydrogen peroxide because old H₂O₂ may give weak or false-negative results. 

3. Add bacterial colony

1.     Using a sterile wooden stick or plastic loop, take a small amount of bacterial colony.

2.     Use a colony from an agar medium without blood.
Blood agar can give false-positive results because blood cells contain catalase.

3.     Mix the colony gently into the drop of H₂O₂.

4. Observe reaction

Observe immediately for bubble formation.

Do not wait too long, because delayed weak bubbling may be nonspecific.

Interpretation

For Clostridium spp., expected result:

No bubbles or very weak reaction = catalase negative.

Controls

If possible, use controls:

D-Indole test using tryptone broth:

1. Medium preparation

Use tryptone broth or peptone water.

Typical tryptone broth composition:

Adjust pH to around 7.2–7.5, then sterilize by autoclaving.

If you already have commercial tryptone broth, prepare it according to the manufacturer’s instructions.

2. Inoculation

1.     Label the tube with the isolate code.

2.     Using a sterile loop, take a small amount of bacterial colony.

3.     Inoculate the tryptone broth.

4.     Mix gently.

For Clostridium spp., the tube should be incubated under anaerobic conditions.

3. Incubation

Incubate at the appropriate temperature for your organism.

For many Clostridium spp.:

35–37°C for 24–48 h under anaerobic conditions

For slow-growing isolates, incubation may need to be longer, depending on your lab protocol.

4. Add Kovac’s reagent

After incubation:

1.     Do not shake the tube strongly.

2.     Add about 0.5 mL Kovac’s reagent to the culture tube.

3.     Let the reagent form a layer at the top.

4.     Wait 1–2 minutes. 

5. Interpretation

Expected result for Clostridium

For Clostridium beijerinckii, indole is generally expected to be:

Indole negative

But this can vary depending on the strain and the identification system, so do not use indole alone to confirm the species.

E-Indole test using SIM medium:

SIM medium tests:

• S: sulfur reduction
• I: indole production
• M: motility

Procedure

1.     Take a tube of sterile SIM medium.

2.     Using a sterile straight needle, pick a colony.

3.     Stab the medium straight down the center.

4.     Incubate at 35–37°C for 24–48 h.

5.     For Clostridium, incubate anaerobically if required.

6.     After incubation, add Kovac’s reagent on top.

7.     Observe for a red ring.

Interpretation

Controls

Use controls if possible:

F-Urease test using Christensen’s urea agar slant:

1. Medium

Use sterile Christensen’s urea agar slant.

The medium usually contains:

The original color is usually yellow-orange/light peach.

2. Inoculation

1.     Label the urea agar slant with the isolate code.

2.     Using a sterile loop, take a fresh bacterial colony.

3.     Streak the surface of the slant.

4.     Do not stab deeply unless your lab protocol asks for it.

5.     Close the tube cap loosely if aerobic incubation is used.

For Clostridium spp., incubation should be under anaerobic conditions if you are testing an anaerobic isolate.

3. Incubation

Incubate at:

35–37°C for 24–48 h

Some weak urease producers may need longer incubation, up to 5–7 days, depending on the lab protocol.

4. Interpretation

G-Urease test using urea broth:

1. Inoculation

1.     Label a sterile urea broth tube.

2.     Inoculate the broth with a fresh colony using a sterile loop.

3.     Mix gently. 

2. Incubation

Incubate at:

35–37°C for 24–48 h

For anaerobes such as Clostridium, incubate anaerobically.

3. Interpretation

Expected result for Clostridium beijerinckii

For Clostridium beijerinckii, urease is generally expected to be:

Urease negative

But results can vary depending on the strain and test system, so urease alone cannot confirm the species.

Controls

H-HYDROLYSIS OF CARBON SOURCES:

1. Prepare basal medium

1.     Prepare the basal broth without carbon source.

2.     Add pH indicator such as phenol red or bromocresol purple.

3.     Adjust pH around 7.0.

4.     Dispense into tubes.

5.     Add Durham tubes if you want to detect gas.

6.     Sterilize by autoclaving. 

2. Prepare carbon source solution

Prepare each carbon source separately, for example:

For heat-sensitive sugars, filter-sterilize and add after autoclaving.

3. Inoculation

1.     Label each tube with the carbon source name.

2.     Inoculate with the bacterial isolate.

3.     Include one negative control tube without carbon source.

4.     Incubate under anaerobic conditions. 

4. Incubation

Incubate at:

35–37°C for 24–72 h

For solventogenic Clostridium, you may need longer incubation, around 48–96 h, especially if you want to detect butanol.

Interpretation

With phenol red

With bromocresol purple

For butanol production specifically

For your C. beijerinckii butanol study, don’t rely only on color change. The best proof is:

Use P2 medium + glucose as the main fermentation medium.

Recommended glucose:

60 g/L glucose for classic butanol production trials.

For 30 mL:

1.8 g glucose

I-GELATIN LIQUEFICATION TEST:

1. Prepare the medium:

1.     Prepare or obtain sterile nutrient gelatin deep tubes.

2.     Allow the medium to solidify before inoculation.

3.     Label each tube with the isolate code. 

2. Inoculation:

1.     Sterilize a straight inoculating needle.

2.     Allow it to cool.

3.     Pick a small amount of bacterial colony.

4.     Stab the needle straight into the center of the gelatin medium.

5.     Withdraw the needle along the same line.

6.     Close the tube.

For Clostridium isolates, incubation should be under anaerobic conditions if required.

3. Incubation:

Incubate at:

35–37°C

Duration:

24–48 h initially, but some organisms need up to 7–14 days.

Gelatin liquefaction can be slow, so if the result is negative after 48 h, continue incubation and check daily.

4. Cooling step:

This step is very important.

After incubation:

1.     Place the tube in the refrigerator or ice bath for about 15–30 minutes.

2.     Then observe whether the medium is solid or liquid.

Why?
At 35–37°C, gelatin may be liquid just because of temperature. Cooling is needed to know if gelatin was truly hydrolyzed.

Interpretation

Expected result for Clostridium beijerinckii

For Clostridium beijerinckii, gelatin liquefaction is often reported as:

Negative or variable depending on strain

So do not rely on this test alone. Use it with Gram stain, endospore stain, catalase, indole, urease, carbohydrate fermentation, anaerobic growth, and ideally 16S rRNA sequencing.

Controls

J-RIFAMPICIN SENSITIVITY:

1. Prepare bacterial suspension:

Prepare a fresh bacterial suspension from a pure culture.

The suspension should be standardized according to your lab protocol, commonly using a McFarland standard.

2. Prepare rifampicin dilutions:

Prepare serial dilutions of rifampicin in sterile medium.

Example range:

0.125, 0.25, 0.5, 1, 2, 4, 8, 16 µg/mL

The exact concentration range should be chosen according to your supervisor’s protocol or CLSI/EUCAST guidance.

3. Add medium and antibiotic:

In each well or tube, add medium containing one rifampicin concentration.

Also prepare:

4. Inoculate:

Add the standardized bacterial suspension to each tube or well.

Mix gently.

5. Incubate anaerobically:

Incubate under anaerobic conditions at:

35–37°C

Incubation time depends on the growth rate of the isolate, but for anaerobes it is commonly around 24–48 h.

6. Read the result:

Check for visible growth/turbidity.

The MIC is:

the lowest rifampicin concentration where no visible bacterial growth is observed.

Example:

Result:

MIC = 1 µg/Ml

K-CURD FORMATION TEST:

A. Litmus milk test procedure

1. Medium:

Use sterile litmus milk tubes.

Commercial litmus milk usually contains:

• skim milk powder
• litmus indicator
• sometimes additional nutrients depending on supplier

The medium is usually purple/lavender before inoculation.

2. Inoculation:

1.     Label the litmus milk tube with isolate code.

2.     Using a sterile loop, take a fresh bacterial culture.

3.     Inoculate the litmus milk tube.

4.     Mix gently, but do not shake strongly.

5.     Close the tube.

For Clostridium isolates, incubate under anaerobic conditions.

3. Incubation:

Incubate at:

35–37°C for 24–48 h

If no visible change appears, continue incubation up to:

5–7 days

Some milk reactions are slow.

4. Observation:

Observe daily for:

• color change
• curd formation
• gas cracks
• clearing
• clot digestion
• reduction of litmus

Interpretation of litmus milk

For some Clostridium spp., a classic reaction is stormy fermentation in milk, where gas disrupts the curd.

B. Skim milk curd formation test

If you do not have litmus milk, you can use sterile skim milk medium.

Skim milk medium example

Sterilization can be done according to your lab protocol, often with careful autoclaving or separate sterilization because milk can caramelize or form precipitates.

Procedure

1.     Prepare sterile skim milk medium in tubes.

2.     Label each tube.

3.     Inoculate with the bacterial isolate.

4.     Incubate anaerobically at 35–37°C.

5.     Observe after 24, 48, 72 h, and up to 7 days if needed.

6.     Look for curd formation, gas cracks, or clearing.

Interpretation in skim milk

L-LUCAS TEST:

1.     Take a clean, dry test tube.

2.     Add about 1 mL of the alcohol sample.

3.     Add about 3 mL of Lucas reagent.

4.     Shake gently.

5.     Keep the tube at room temperature.

6.     Observe the time needed for cloudiness/turbidity to appear.

Interpretation

Explanation

Tertiary alcohol

Reacts very fast because it forms a stable tertiary carbocation.

Example:

tert-butanol → immediate turbidity

Secondary alcohol

Reacts more slowly.

Example:

isopropanol → turbidity after a few minutes

Primary alcohol

Usually does not react at room temperature.

Example:

ethanol → no turbidity or very slow reaction

3-