Lactobacillus - Science topic

doi: 10.1080/00218839.2020.1746019

Many scientific genetic analyses ran in the past few years have shown that the huge heterogeneity. it no longer reasonable to group all these 261 species under the genus Lactobacillus

Dear Christos Velesiotis

First, the OD values you shared are too high. Ideally you should try to adjust OD of your non-treated samples to 0.7-1.2 range for an MTT assay. You may increase the volume of isopropanol but I think you might just have too many cells to begin with. Therefore another strategy would be to decrease the number of cells you seeded. I know this does not answer your question exactly but at high absorbance levels, you cannot compare two values with confidence.

Second, the cells may go through growth arrest via contact inhibition, if cell density is too high. It may even induce apoptosis. This may explain having lower OD for 72h non-treated cells. Both problems can be avoided by seeding fewer cells.

Cheers

The reason that MacConkey is selective for gram negative is the use of bile salts and crystal violet. So in theory you could add those two agents to your media (in other words make a TSA based version of MacConkey). the suggestion of Robert Adolf Brinzer is also good. I think most gram - are fairly tolerant to vancomycin. However I would not suggest beta-lactams, those are still quite inhibitory to gram - bugs but just at higher concentrations in general.

Hi Paul,

I checked for Lactobacillus and found that 16S have 1.5 kb and 23S have around 3kb.

Thanks

I took this direct from one of the directions for using MRS agar:

"Lactobacilli MRS Agar is used for the cultivation of lactobacilli and is not intended for use in the diagnosis of disease or other conditions in humans. MRS Agar is a medium for the cultivation and enumeration of Lactobacillus spp. Originally developed in 1960 by de Man, Rogosa & Sharpe, the medium is suitable for most lactic acid bacteria and is intended as a substitute for Tomato Juice Agar. When acidified to pH 5.4 MRS Agar can be used to enumerate Lactobacillus bulgaricus in yogurts. Nutrition is provided by a mixture of carefully selected peptones, glucose, beef & yeast extracts while Tween® 80, magnesium and manganese sulfates act as growth stimulants. Selectivity against streptococci & molds is provided by ammonium citrate and sodium acetate. Used at low pH, ammonium citrate allows growth of lactobacilli while inhibiting a number of other microorganism groups. Occasionally, sterilization of this medium at 121°C for 15 minutes, in some autoclaves, may cause the pH to fall outside of the specified pH limits 6.4 +/- 0.2. In these rare cases, adjustment of the medium using acetic acid or sodium hydroxide is recommended."

As you can see taking out sodium acetate will leave ammonium citrate in there as a selective agent. You would have to take both of them out but you then risk getting moulds. Considering the pKa of lactic acid to be about 3.8. I would think using a medium like Muller Hinton but adjusting the pH to about 4.5 and temperature 40 to 45 degrees would select for lactic acid bacteria.

Thanks Junjie Shao - yes the plasmid is specifically for lactobacillus and contains a constitutive promoter so the gene should transcribe.

Thanks Adam B Shapiro - I will have a look at that database

Optimum temperature for Lactobacillus is 37 degree Celsius while for majority of fungus is 25 degree Celsius meaning a natural fungal barrier. While anti-fungals may affect the gene profile of Lactobacillus strains.

Gordana Zavisic

Thank you very much for the observations!

Normally, I leave the Petri dishes with the culture in the fridge for a maximum of 3 to 5 days.

Previously, I already tried using oil as you advise me and it grows well. Even without using oil and filling the test tubes to the top with MRS medium it gives good growth.

Interestingly, the problem occurs when trying to subculture from the previous grow. I have not found information on whether it requires special handling conditions.

I haven't tried incubating the tubes without shaking as you mention, I'll try that option.

Thank you again!

el proceso de síntesis se lleva a cabo siempre y cuando haya producción de enzimas descarboxilazas por las bacterias acidolácticas

I found these links, kindly check them, they may be useful:

Please read this article

Depends on a lot of factors and what exactly you are looking for. Any sample introduced into GCMS, needs to be thermally stable and also volatilized inorder to be separated on the GC column for efficient identification on the MS. If it is volatiles (VOCs) you are looking for, you may not need any further processing of the samples. If you are looking for lipids, fatty acids, esters and likewise compounds, some sort of pre-processing may be required, like derivitization techniques - acylation, alkylation, silylation, among others.

In my opinion the appropriate atmosphere should be used to provide ideal anaerobic conditions for growth, because I had this problem when I isolated Campylobacter jejune as it is a microaerophile, so I used its own atmosphere packs with an anaerobic jar because it is tightly covered but sometimes when something went wrong and I open the jar for a certain reason the colony growth on the next day is not satisfactory and they are small, so using its appropriate atmosphere with an anaerobic jar to get the satisfactory growth of the colonies may be appropriate solution... Best wishes in your work

Microencapsulation is the best remedial for controlled release of pH in any food matrix. Moreover this method is also effective to maintain the viability of probiotics for longer time.

From a past experience, antimicrobial peptides and proteins should precipitate with 70-75% saturation of ammonium sulphate.

Ammonium sulphate precipitation of the cell-free supernatant will produce for sure brown pellets that contain most of the bacteriocins (if any).

You can then dissolve your pellets with PBS or water then desalt on Sephadex or any other method (Vivaspin, dialysis, c18 cartridges), then test for inhibitory activity.

This is the ultimate proof that you have proteinaceous antimicrobial substances that are worth further mass spectrometry analysis.

Quite unlikely to succeed..making 108 cfu is a different ball game....

The Proteins can be concentrated by precipitation from solution with ammonium sulfate, polyethylene glycol, organic solvent, trichloroacetic acid, potassium chloride/sodium dodecyl sulfate, and three-phase partitionic

The media most commonly used for cultivation of lactobacilli are de Man Rogosa Sharpe, Lactobacilli selective agar and Rogosa agar.

Hello Dr Allison Pham

Best regards

It is normal to grow Candida albicans cells in 5% CO2 together with human cells, such as macrophages or those in reconstituted epithelia. Hence, there should be no problem for both to live together.

I hope this will help

I use the protocol suggested in the NICE manual and it works great

https://www.mobitec.com/media/mobitec/category-preview/vector-systems/pdf/3/NICE_Expression_System-Handbook.pdf

Dear Dr Imaniar Imaniar,

I think the protocols found in these articles (attached) may actually help you, since researchers are using GC-MS approach for the characterization of Lactobacillus and other microbial strains in dairy products.

Best wishes,

Sabri

Maybe these references can help you.

- Song X. et al. (2017). CRISPR-Cas9 D10A Nickase-Assisted Genome Editing in Lactobacillus casei. 10.1128/AEM.01259-17

- Gury J. et al. (2004). Random transposon mutagenesis of Lactobacillus plantarum by using the pGh9:ISS1 vector to clone genes involved in the regulation of phenolic acid metabolism. 10.1007/s00203-004-0705-1

- Licandro-Seraut H. et al. (2012). Development of an efficient in vivo system (Pjunc-TpaseIS1223) for random transposon mutagenesis of Lactobacillus casei. 10.1128/AEM.00531-12

Thanks for your answers!!!

If I get it right, it seems that the CytoTrace (CMTPX) work fine while the acetylated fluorophores like fluorescein diacetate and other lipophilic fluorophores do not. Due to the fundamentally different structures, I would therefore suggest to try other fluorophores based on the CytoTrace technology.

Also check https://www.researchgate.net/figure/Effect-of-pH-and-temperature-on-the-antimicrobial-activity-of-the-supernatant-from-L_tbl1_263403611/amp

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interested

plz. check the attached file

Check https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0145784

This could be of help

Lactobacillus is fastidious bacteria. So I suggest to use MRS L- cysteine.HCl medium. It's modified MRS broth medium by adding L- cysteine.HCl to be anaerobic medium. It's a rich media you can use it for others species. Or you can use modified Nutrient broth media by adding sucrose (as a carbon source) and L - cysteine.HCl (as an anaerobic factor).

We use MRS for a general Lactobacillus growth.

I totally agree with Dr. Alaa Kareem Niamah as molecular identifications are very accurate and cannot be wrong because they are designed as genetic maps of microorganisms. But there are rare cases in which it gives negative results and not different and this is for several reasons (the isolates may have been isolated from food materials and some of the components of the food affected the sensitivity of the kit, or sometimes the concentration of the bacterial broth may be less than the sensitivity of the kit, Sometimes, and in rare cases, mutations occur in the bacteria that make them resistant to heating also affect the sensitivity of the kit), but these cases are recorded for special types of bacteria, and they rarely occur.

Hi,

See in attach file

Also check https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155122

Optimal initial pH value for the growth is in the range from 6.4 to 4.5. It grows as rods, single rods or in short chains.

Mohammad Afzal Hossain Thank you so much for your suggestions.

Agree with you sir @Jacobsimons

Also check https://www.eppendorf.com/product-media/doc/en/809547/Fermentors-Bioreactors_Application-Note_412_BioFlo-120_320_Microaerobic-Fermentation-Lactobacillus-acidophilus-within-Gut-Microbiome-Physiological-Conditions-BioFlo-Bioprocess-Control-Sta.pdf

Also check https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5428095/

Dear Simran Makan 16S rRNA and Shotgun sequencing are two different things, please do an extensive research about this, then you will have a better clue of the next thing pls

Thanks Marilia Cavalcanti

I wouldn't be an expert in bacterial molecular work but from those details I would expect a high variation between samples. So many variables we cannot account for. If you can figure out a standardisation method for calculation that might be the best thing. Maybe something like ct/no. of CFU on the final streaks etc. Defiantly a topic to chat with others in your lab about.

Best of luck

First, since Lactobacillus acidophilus is a Lactobacillus, you cannot isolate it from itself. Second, a good scholar.google search on methods would help find a variety of choices. The following is not specific for L. acidophilus but it shows the ability to discriminate strains of Lactobacillus from each other, and then of course you have the ability to isolate a colony -

Good question

PFA is a crosslinking reagent! You have cross-linked your bacteria forming a networked gel/sponge. They will not sediment at 10,000 g in 5b minutes!

Suggest that you use cold methanol, or similar, to fix your bugs.

Hi Xiangang

It does not seem to be logically necessary to choose exactly 595nm. For optical densities you are measuring a transmission in a turbid solution not an extinction.

You avoid wavelenghts close to the UV spectra to filter out results from dissolved molecules and blank against your medium to eliminate VIS-Colours in the samples

If there is a possible maximum extinction of undissolved by-molecules that have maximum of exactly 595nm, they will be already visible at 590nm or 600nm.

You have to take in mind too, that a linear progression of the results for turbid solutions is only given up to arround ext. 0,7, above this value it is usefull to dilute.

Possible reasons might also be: Perhaps researchers had good experiences with this wavelenght.. or used instruments with exactly this (fixed) filter.

Anyway: the value for ODs can differ between single photometers it is not 100% consistent, if you want to exactly estimate the rate of living cells, you should perform a living cell count, f.e. via plating.

Theodorus Eko Pramudito you need to check your universal primer wich phylum cover. I'm curious if you resolve the problem.

MRS medium is rich in minerals and growth factors and hence is used for such fastidious lactobacilli.However, it could be replaced successfuly by a media based on whey enriched with minerals in many studies,

limiting nitrites in dairy food is highlighted in food safety research. Several researchers have found that inoculation of Lactobacillus sp.could inhibit accumulation of high concentrations of nitrites during fermentation since they have enzymes to degrade nitrite.

L. paracasei is genotypically and phenotypically indistinguishable from other members of its genus such as Lactobacillus casei and Lactobacillus rhamnosus

The precise wavelength is not that important because you are not measuring absorbance, only turbidity. The relationship between the OD and the cell density will be steeper at shorter wavelengths, resulting in somewhat greater sensitivity. Whatever wavelength you choose, you should calibrate the measurement by plating the cell culture at various ODs and measuring the colony counts.

There's typically three separate issues with regards to moving genes between firmicutes (low %GC gram positives like lactobacilli) and E. coli

1. Plasmid origin recognition. Most E. coli origins don't replicate in firmicutes, and many broad host range gram positive plasmid origins (ie. pAMβ1) don't work in E. coli. The only exception I know of is the pWV01 ori, which can replicate in almost all species that have been tested (G+ and G-), but that only replicates fine in recA+ hosts, so it won't transform well into most cloning E. coli strains. This is why most gram positive vectors available are shuttle vectors with a G+ origin and a separate E. coli origin of replication.

2. Promoters. E. coli has pretty sloppy promoter recognition requirements to initiate transcription, firmicutes tend to be pretty strict. As a result, most sigma 70 promoters from firmicutes will work in E. coli (sometimes too well) but E. coli promoters almost never work in firmicutes. The ribosome binding sites for initiating translation can be a bit different too, but again that's mainly a problem when taking E. coli ribosome binding sites and moving them to a G+, not usually vice-versa. When in doubt, always use an established promoter + RBS known to work in both organisms for gene expression.

3. Codon usage. Firmicutes, being low %GC, can have very different codon biases compared to E. coli. This is mainly an issue if you need high level expression of a particular protein. Just be aware that most coding regions from a G+ will probably have a significant amount of rare E. coli codons and vice-versa.

All that being said, researchers use E. coli-G+ shuttle vectors all the time, you just have to be aware of which components will work in both organisms and which won't.

Why don't you check a few of the red colonies to see if they carry the RFP gene on the plasmid? It could be that most of your plasmid was correct but some small fraction was original and still has RFP. The other possibility is that the red colonies are not lactobacillus at all but some red contaminant like Serratia which is pretty resistant to most antibiotics.

Both are same. Bacillus coagulans was previously known as Lactobacillus sporogenes.

Thank you very much for all your answers. I will apply the experiment you mentioned as soon as possible.

Best regards..

Thanks everyone for your responses.

Hello, Nice paper of prediction of competitive microbial growth in mixed culture is attached.

Good luck!

Hi,

The MRS uses for Lactobacillus isolation from different sources. But enterococcus isolation should be anther media such as M17 or Soduim Azide Agar.

I ready for any help.

MRS agar normally used to isolate lactobacilus or all type of LAB, you can add specific nutrients if you want to isolate specific LAB, calcium carbonate can be added before autoclaving the media, but I usually add bromocresol purple into media, which is the same thing.. regards

Thank you Dr. Carole. Even I was looking for a reference on the same topic.

Hi, Lala K Nagaraj,

Thanks for your reply YPG medium is Yeast extracr, peptone & and glucose are the main components, besides other minor ones. Good luck.

Past studies, Soy milk media is good media for Lactobacillus spp. compared with Bifidobacterium spp.

Hi,

You can use promega kit. I used it.

I have this strain but I am from Iraq.

I've had good luck with the longer "gram negative bacteria" protocol with the Qiagen kit. It uses lysozyme and a longer heat step. Works well even in the hands of undergrads.

Hello, APi test is better to use after fulfilment culture belongs to LAB or not. As I see the question was differentiation of LAB from other Bacilli. API test is for identification of Lactobacilli in subsipecies level. The use of API test just for differentiation LAB from other bacilli is very expensive and time wasting way.

Hi,

You can use whey which media complete and it is not add any compound.

It depends to some degree on the intended end use of your genomic DNA preparation. For example, if it is for direct hybridisation, you will want ample quantities of unsheared gDNA. But if it for use as a template involving an amplification step, that is generally more forgiving. The issue may be the gDNA isolation protocol, but without knowing the specific details of the protocol being used or the end use of the gDNA, it is difficult to identify where in the process the problem is occurring.

No doubt it might help but many a times a dye like crystal violet can be inhibiting.

It's well-known that in several species of LAB, the terminal d-alanine residue is replaced by d-lactate or d-serine in the muramylpentapeptide, preventing vancomycin binding and therefore becoming resistant to this antibiotic.

Regards

The initial screening and selection of probiotics includes testing of the following important criteria: 1.phenotype and genotype stability, including plasmid stability; 2.carbohydrate and protein utilization patterns;

3.survival and growth intestinal epithelial adhesion properties;

4. production of protease resistant bicteriocines;

5. acid tolerance

6. bile tolerance

7. antioxidant activity

also

voluble for industry Inuinase activity, betta calactosidase activity.

And very voluble if you can find sporolactobacillus - sporoforming lactobacilly with any above mentioned activities.

try to reanimate the culture of Lactobacillus by co-incubating them with the culture fluid or autoclaved cells of the susceptible strain